Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice

Breast cancer resistance protein (Bcrp/Abcg2) is a member of the ABC transporter family. The purpose of this study was to quantify Bcrp mRNA in rat and mouse tissues, and to determine whether there are gender differences in Bcrp mRNA expression. Rat Bcrp mRNA levels were high in intestine and male kidney, and intermediate in testes. Mouse Bcrp expression was highest in kidney, followed by liver, ileum, and testes. Male-predominant expression of Bcrp was observed in rat kidney and mouse liver. Furthermore, gonadectomy and hypophysectomy experiments were conducted to determine whether sex steroids and/or growth hormone are responsible for Bcrp gender-divergent expression patterns. Male-predominant expression of Bcrp in rat kidney appears to be due to the suppressive effect of estradiol, and male-predominant expression of Bcrp in mouse liver appears to be due to the inductive effect of testosterone.     

Distribution and regulation of rat insulin-like growth factor I messenger ribonucleic acids encoding alternative carboxyterminal E-peptides: evidence for differential processing and regulation in liver

Alternative splicing of insulin-like growth factor I (IGF-I)/somatomedin C mRNAs generates two IGF-I mRNAs coding for IGF-I peptides with different sequences in the E domain of the IGF-I prohormone. These two mRNAs encode alternative E peptides due to the presence (IGF-Ib) or absence (IGF-Ia) of a 52-base insert in the region coding for the E domain. We have used a solution hybridization/RNase protection assay to determine the tissue distribution and regulation by GH of the expression of these alternative IGF-I mRNAs. IGF-Ib mRNAs are present in low abundance (representing approximately 2.5% of the total IGF-I mRNA) in heart, lung, muscle, testes, stomach, kidney, and brain, but represent approximately 13% of the IGF-I mRNA in liver. GH treatment of hypophysectomized rats increased steady-state IGF-I mRNA levels in liver, kidney, lung, and heart. In kidney, lung, and heart, IGF-Ia and IGF-Ib mRNA levels were coordinately regulated by GH, but, in liver, the fold increase in IGF-Ib mRNA levels was approximately three times greater than the fold increase in IGF-Ia mRNA levels. These data suggest that the processing of IGF-I mRNA in liver is different than in nonhepatic tissues. These results also further elucidate the organization of the rat IGF-I gene as well as the generation of multiple IGF-I mRNAs by alternative splicing.     

Melatonin and Environmental Lighting Regulate ALA‐S Gene Expression and So Porphyrin Biosynthesis in the Rat Harderian Gland

The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day‐night PPIX levels as well as day‐night activity and mode of expression of the porphyrinogenic enzymes δ‐aminolevulinate synthase (ALA‐S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630‐ to 670‐fold higher than in the liver, without a day‐night difference, which is the consequence of both low Fech gene expression (5‐ to 7‐fold lower than in the liver) and ALA‐S over‐expression (4‐ to 7‐fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day‐night variation, whereas HG ALA‐S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA‐S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day‐night ALA‐S variation in the HG that is observed under standard 12 L∶12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA‐S gene expression in the rat HG.     

Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver

We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.     

An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization.

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.     

Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues.

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.     

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Reduced expression and increased CpG dinucleotide methylation of the rat APOBEC-1 promoter in transgenic rabbits

Editing of apolipoprotein (apo) B mRNA in liver limits the plasma LDL levels in horses, dogs, rats or mice. Species such as man or rabbit do not edit the hepatic apo B mRNA and are therefore susceptible to atherosclerosis and coronary artery disease due to elevated plasma LDL levels. The catalytic subunit APOBEC-1 is the only missing component of the apo B mRNA editing enzyme complex in the human or rabbit liver. Here we describe the generation of transgenic rabbits in which APOBEC-1 expression is mediated by the proximal promoter of the rat APOBEC-1 gene. These transgenic rabbits are healthy and fertile, and rat APOBEC-1 mRNA is expressed in liver, intestine, kidney, lung, brain and muscle. The transgenic APOBEC-1 expression is low and not sufficient to induce editing in rabbit liver. In rat, the proximal APOBEC-1 promoter demonstrates a progressive loss of CpG dinucleotide methylation towards the core promoter region that is entirely unmethylated. In the transgenic rabbits, this distinct pattern of CpG methylation is lost, and throughout the entire rat APOBEC-1 promoter, >90% of the CpGs are methylated. Thus, the weak proximal rat APOBEC-1 promoter appears to be down-regulated in the rabbit and may be species-specific.     

Knockdown of legumain inhibits cleavage of annexin A2 in the mouse kidney

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro. In this study, to determine whether annexin A2 is cleaved by legumain in vivo, siRNA-lipoplex targeting mouse legumain was injected into mouse tail veins. Mouse kidneys were then isolated and the effect of knockdown of legumain expression on annexin A2 cleavage was examined. The results showed that both legumain mRNA and protein expression levels were decreased in the siRNA-treated mouse kidneys and that legumain activity toward a synthetic substrate, Z-Ala-Ala-Asn-MCA, was decreased by about 40% in the kidney but not in the liver or spleen. Furthermore, cleavage of annexin A2 at the N-terminal region was decreased in the mouse kidney that had been treated with the legumain siRNA-lipoplex. These results suggest that legumain siRNA was delivered to the kidney by using LipoTrust and that the reduced legumain expression inhibited legumain-induced degradation of annexin A2 in vivo.     

Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration

The molecular cloning of the cDNA coding for a Ca2+-binding proteinregucalcin and its mRNA expression in mouse liver were investigated. ThecDNA clone encoding a regucalcin was isolated from a mouse liver cDNAlibrary and sequenced. Analysis of the sequence of the cloned cDNA showedthat the cDNA encoded the complete amino acid sequence of the mouseregucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouseregucalcin was composed of 299 amino acid residues, and its molecular weightwas estimated to be 33,406 Da. The amino acid sequence of mouse regucalcinhad 94% homology, as compared with that of rat regucalcin. Northernblot analysis with the mouse liver cDNA probe revealed that mouse regucalcinmRNA was mainly present in the liver but only slightly in the kidney with asize of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher thanthat of female mouse. A single intraperitoneal administration of calciumchloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced aremarkable increase in regucalcin mRNA in the liver; the increase inregucalcin mRNA levels at 30 min after calcium administration wasdose-dependent. The present results demonstrate that regucalcin mRNA in miceis uniquely expressed in the liver, and that its expression is stimulated bycalcium administration.     

Prenatal and postnatal expression of mRNA coding for rat prothrombin.

The levels of prothrombin mRNA in prenatal and postnatal rat tissues were analyzed in order to determine tissue distribution of prothrombin expression and to determine if increases in liver prothrombin mRNA during development correlated with previously documented developmental increases in plasma prothrombin levels. Maternal tissues were also analyzed in order to determine if prothrombin mRNA levels varied due to gestational or postpartum influences. Northern analysis demonstrated that rat liver prothrombin mRNA levels increased several-fold late in gestation and reached maximal levels by 13 days after birth. Prothrombin mRNA was also expressed in diaphragm, stomach, intestine, kidney, spleen and adrenal tissues during development. In maternal tissues during pregnancy, prothrombin mRNA was expressed in liver, diaphragm, stomach, uterus and placenta. Prothrombin mRNA levels in each of these tissues that were positive by Northern analysis were quantitated by solution hybridization analysis. Between gestational day 18 and postnatal day 13, liver prothrombin mRNA levels increased from approx. 600 to 2100 molecules per cell (a 3.5-fold increase). In maternal liver during pregnancy, between day 18 and day 22, prothrombin mRNA levels increased from approx. 1800 to 2100 molecules per cell. Immediately after delivery, maternal liver prothrombin mRNA levels decreased to approx. 50% of preparturition levels. Prothrombin mRNA levels in placental tissue ranged from approx. 100 to 250 molecules per cell. In other fetal, postnatal and maternal tissues, prothrombin mRNA expression was less than 100 molecules per cell. These results demonstrate that the level and tissue-type expression of prothrombin mRNA varies in response to prenatal and postnatal influences.