In the group of apoptosis-related genes, two general trends were noted in table 3. (1) The number of genes with varied mRNA levels was always higher in male mutant mice than in female mutant mice. In 6 month old mice, 11 apoptosis-associated genes had altered mRNA levels in male mutant mice while in females; only 6 apoptosis genes had changed expression levels. In 24 month old mice, 10 apoptosis-associated genes had significant changes in mRNA levels in male mutant mice; while in females, the number decreased to 5. (2) Most anti-apoptosis genes had increased mRNA levels; on the contrary, most pro-apoptosis genes had decreased expression levels in due to RXRα deficiency. In male mice, all 4 anti-apoptosis genes in 6 month old mice and 4 out of 5 anti-apoptosis genes in 24 month old mice had increased mRNA levels. On the other hand, 4 out of 7 and 1 out of 1 pro-apoptosis genes decreased in mRNA expression level, respectively, in 6 and 24 month old male mice. The same trend was also found in female mutant mice. A combination effect of up-regulated anti-apoptosis genes and down-regulated pro-apoptosis genes indicated that RXRα-deficient hepatocytes have a more resistant capacity to apoptosis and that hepatocyte RXRα deficiency might have a pro-survival effect.

Genes in this group play important roles in cell migration and angiogenesis and are associated with metastasis, a key feature of malignant cancer cells. Generally, the trends observed in this group were different depending upon age and gender (table 4).

Several HSP (Heat Shock Protein) genes decreased their mRNA levels due to RXRα deficiency (table 5). At 6 month of age, male and female mutant mice had 6 and 2 HSP genes with decreased mRNA levels, respectively. At 24 month of age, two HSP genes, HSPB1 (heat shock 27 kDa protein 1) and HYOU1 (hypoxia up-regulated 1), showed decreased mRNA levels due to RXRα deficiency.

The HSP gene family is highly conserved in structure from C. elegans to humans. HSP genes constitute the cellular protection mechanism and can be induced by various physical, chemical, and biological factors. In 6 month old RXRα-deficient male mice, DNAJB1 (Dnaj homologue, subfamily B, member 1), HSPB1, HSPA1A (Heat Shock 70 KDa Protein 1A), and HSPA1B (Heat Shock 70 KDa Protein 1B) had reduced mRNA levels by 11.30-, 10.29-, 3.86-, and 3.42-fold, respectively. This coordinated down-regulation of the HSP family genes indicated that these genes were regulated by common mechanisms. In 6 month old hepatocyte RXRα-deficient female mice, only 2 genes (HSPA8 and HSPB1) had reduced mRNA levels of 2.50- and 4.66-fold, respectively. Thus, hepatocyte RXRα deficiency has a greater impact on HSP gene expression in male than in female mice. In aged mice, there was no gender difference in the expression pattern of the HSP family genes related to RXRα deficiency. HSPB1 and HYOU1 both exhibited decreased mRNA levels in male and female aged mutant mice to a similar extent. HSPB1 mRNA levels were consistently decreased in both male and female mutant mice of both age groups.

Some small HSP genes expression levels such as HSP27 can be up-regulated by RXR/RAR heterodimer in lens 22; furthermore, RXR ligand 9-cis RA (retinoid acid) increases the HSP gene expression in Jurkat cells 23. Rocchi, P., et al. suggested that the expression of HSPB1 could be up-regulated by androgen ablation 24. Another report revealed that exogenous androgen treatment could delay stress response by decreasing the expression of HSP70 25. Down-regulation of these HSP genes implied that RXRα-deficient hepatocytes had a reduced protective ability and might be more susceptible to injury resulted from external stimuli compared with wild type hepatocytes. It is possible that there was increased androgen signaling activity due to RXRα deficiency because RXRα is a negative regulator for the androgen pathway, leading to inhibition of HSP family mRNA expression. Another phenomenon that we observed is that the number of HSP family genes which showed alteration in mRNA levels is higher in male than in female when mice are 6 month old. However, this gap decreased when mice were aged. Since androgen levels are higher in male than in female and decrease during aging, the physiological changes of androgen levels may account for this gender- and age-dependent gene expression pattern. It has been shown that RXRα-deficient hepatocytes have a shortened lifespan 6. Our data implied down-regulation of HSP genes expression in RXRα-deficient hepatocytes might result in decreased cell protection ability and consequently render the cells prone to death.

Retinoids could arrest cell cycle progression and induce apoptosis in many types of cancer cells through activation of RXRs. RXRα signaling plays critical roles in cell growth and differentiation. In RXRα hepatocyte-deficient mice, many genes associated with cell growth had changes in their mRNA levels. Again, this difference in gene expression pattern is gender and age dependent (table 6).

More genes altered expression patterns in male mutant mice compared with female mutant mice at 6 month of age (both in numbers and fold). For example, Jun, Fos, and Myc mRNA levels decreased by 2.87-, 5.74-, and 7.76-fold, respectively, in male mutant mice; on the other hand, tumor suppressor genes such as KLF6 (Kruppel-like factor 6), EGR2, and EGR1 (early growth response 2 and 1) were down-regulated by 5.67-, 6.03-, and 15.88-fold, respectively. KLF6 inhibits cell proliferation through up-regulation of p21 expression in liver cells 26. EGR1 and 2, early transcription factors, increase apoptosis in tumor cells. The down-regulation of oncogenes indicated that in RXRα-deficient hepatocytes, the cell growth activity was compromised, providing another reason for the observed shortened cell lifespan due to RXRα deficiency. The same trend observed in tumor suppressor genes suggested that negative regulation of cell cycle was also impaired. For example, p21 is up-regulated by activation of RXR/RAR dimer in HepG2 cells 27. Consistently, our data showed the decrease of p21 mRNA levels by 2.32 folds in 6 month old male mutant mice. BTG2 (B-cell translocation gene 2), a downstream effector of the p53 pathway 28, also had a 5.26 fold reduction. Several genes belonging to the p53 pathway had altered mRNA levels and lead to compromised p53 pathway activity. The impairment of the p53 pathway and other negative regulators implied that due to RXRα deficiency hepatocytes would surpass the cell cycle barrier more easily and be transformed into malignant cells. In female mutant mice, the mRNA levels of the above mentioned genes were not changed. One obvious trend the data points to is that all negative regulator genes for cell cycle transition decreased their mRNA levels in female mutant mice. It also implies cell cycle checkpoint machinery is impaired in female mutant mice.

Contrary to 6 month old female mutant mice, 24 month old female mutant mice had more genes with modified mRNA levels than did the male mutant mice of the same age group (22 vs. 10). In addition, no obvious trends in gene expression patterns were noted. Oncogenes and tumor suppressor genes were up or down regulated randomly.

The impact of RXR-mediated pathways on cell growth is very complex. It can be tissue- or cell type-specific. The activation of RXR/CAR, RXR/PXR, and RXR/PPARα pathways could induce hepatomegaly 293031. On the contrary, RXR/RAR or RXR/VDR pathways inhibit tumor cell growth 132. It is likely that the complexity of changes seen in gene expression profiles reflect the net results of combined proliferative and anti-proliferative effects due to hepatocyte RXRα deficiency.

Our data implied that in matured livers, RXRα deficiency has more impact on cell growth-related gene expression levels in males than in females; but in aged liver, female mice have more changes in cell growth-related gene expression patterns than do male mice.

Immune response-related genes also had significant changes in mRNA level due to RXRα deficiency (table 7). At 6 month of age, male and female mutant mice had 15 and 4 genes with modified mRNA levels, respectively; however, at 24 month of age, there were 10 and 24 genes with change expression levels in male and female mice, respectively. In aged mice, the number of genes with altered mRNA levels increased significantly in female mutant mice. Another striking change in aged mice was that most of the immune response-related genes (8 out of 10 in males and 18 out of 24 in females) increased in mRNA levels. Such expression trends were not found in 6 month old mice, implying increased inflammation status might occur in both genders at old age due to RXRα deficiency.

The RXR/PPAR dimer attenuates the inflammation response in colon 32. RXR and PPAR agonists decrease TNFα (tumor necrosis factor α) and IL-1β (interleukin 1β) mRNA levels. In liver tissue, the acute response to external stimulus was associated with a down-regulation of RXRα expression 33. Lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRα, β, and γ proteins in hamster liver 33. Alcohol induced pro-inflammation gene expression (TNFα, IL6, and IL1β) is enhanced due to hepatocyte RXRα deficiency 34. These observations implied an inverse correlation between inflammation and RXRα signaling. These findings indicate that RXRα deficiency increases inflammation response to stimulus, which might be due to deregulation of a panel of immune-related genes. Furthermore, the impact of RXRα deficiency on immune response genes was more obvious in aged than in young mice.

The expression of many hepatic genes are gender dependent 35. However, the findings might vary depending upon the age and strain of mice studied. The susceptibility of night blindness and xerophthalmia, the most common symptoms of vitamin A deficiency, are also gender dependent; the incidence is higher in males than females 36. Thus, we studied the impact of RXRα deficiency on the expression of hepatic cancer-related genes that have a gender-dependent expression pattern. Our data showed that the numbers of gender-dependent genes in 6 month old wild type and hepatocyte RXRα-deficient mice are 329 and 200, respectively. When the mice were aged, the number of gender-dependent genes in wild type mice dropped significantly (127), but not so much in mutant mice (167) suggesting aging narrowed the gender gap more in wild type mice than in the mutant mice (table 8). There were common gender-dependent genes in both wild type and hepatocyte RXRα-deficient mice. The names of the genes that showed the greatest fold changes and those genes had the greatest fold changes due to RXRα deficiency at 6-month old age are listed (tables 9 and 10). Also at 24-month old, those genes showed the greatest fold change and genes had the greatest fold changes due to hepatocyte RXRα deficiency are listed in Tables 11 and 12. Surprisingly, the gender-dependent hepatic gene expression was also age-dependent as there was no overlap between the two age groups. Our data indicate that RXRα deficiency affects gender-dependent hepatic gene expression and that this effect is age-dependent.

Animal protocols were approved by the institutional animal use committee of the University of Kansas Medical Center, Kansas City. Male and Female C57BL/6J wild type and RXRα knock out mice were weaned at 28 days, housed individually, given free access to water, and randomly assigned to study groups. Four groups of mice were used to determine the effects on gene expression profile at two ages in both male and female mice. Each group had 3 mice. At particular time points after birth, 6 month (mature) and 24 month (aged) mice were sacrificed by cervical dislocation, and the livers were rapidly excised and flash frozen in liquid nitrogen. No signs of pathology were detected in any of the animals used.

Total RNA was isolated from frozen mouse livers using Trizol Reagent (Invitrogen Corporation, Carlsbad, CA) and further purified using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Total RNA was quantified by UV spectrophotometry and its integrity and quality were assessed on RNA 6000 Nano LabChips with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Total RNA was reverse transcribed into cDNA using reverse transcription kit provided by Invitrogen Company. Synthesis and purification of double-stranded cDNA were conducted as described in the Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Biotin-labeled cRNA was synthesized from the purified cDNA using the BioArray High Yield Transcript Labeling Kit according to the manufacturer's specifications (ENZO Life Sciences, Farmingdale, NY). Labeled cRNA was purified using the GeneChip Sample Cleanup Module, quantified by UV spectrophotometry and assessed for quality with the Bioanalyzer 2100. Twenty μg purified cRNA was fragmented and 15 μg fragmented cRNA was hybridized to Affymetrix Mouse Genome 430A 2.0 Array GeneChips (Affymetrix, Santa Clara, CA) according to the Expression Analysis Technical Manual. Washing and staining of the hybridized arrays were carried out using the Fluidics Station 400 and arrays were subsequently scanned with the Hewlett Packard GeneArray Scanner.

Affymetrix scan data (.cel files) were imported into Rosetta Resolver for analysis (Rosetta Biosoftware, Seattle, WA). Following intrachip normalization and background correction, individual replicates were combined into single "ratio experiments" by an error-weighted method using the control group as a baseline. An agglomerative hierarchical clustering algorithm utilizing an error-weighted Euclidian distance measure was performed on the ratio experiments to identify active genes. Transcripts were defined as active if they increased or decreased by greater than 2.0-fold. The microarray data from this work was submitted to the ArrayExpress database and the accession number is E-MEXP-1711.

The synthesized cDNA was diluted 20 fold by water. All primers and probes were designed based on nucleotide sequences in Genbank using the Primer Express software (PE Applied Biosystems). PCR reaction efficiency was calculated for each primer pair using with five dilution points of the calibrator sample to validate primers and probes. The PCR product covered at least two exons according to introns-exons organisation of selected genes. Each real-time PCR reaction consisted of 1× PCR Master Mix (PE Applied Biosystems), 0.5 μM forward and reverse primers and 1 uM corresponding probe. Final volume is 20 μl. Reactions were carried out on ABI PRISM 7900 real time PCR system (PE Applied Biosystems) for 40 cycles (95°C for 15 s, 60°C for 1 min). The expression fold change for each gene was calculated using the Ct method and β-actin was used as an internal control.