Regucalcin mRNA Levels Research Articles

Nuclear localization of regucalcin is enhanced in culture with protein kinase C activation in cloned normal rat kidney proximal tubular epithelial NRK52E cells

In this study we investigated whether the nuclear localization of regucalcin in cloned normal rat kidney tubular epithelial NRK52E cells is regulated after culture with hormonal signaling factors. Stable regucalcin/pCXN2 transfectants with subconfluent monolayers were further cultured for 24 or 48 h in a serum-free medium containing either vehicle, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), parathyroid hormone (PTH), phorbol 12-myristate 13-acetate (PMA), or other factors. Culture with TNF-alpha (1.0 ng/ml of medium) or TGF-beta1 (5.0 ng/ml) for 48 h caused a significant decrease in regucalcin mRNA levels in NRK52E cells (wild-type), while regucalcin mRNA levels were markedly increased in the presence of PMA (10(-6) M), an activator of protein kinase C, in wild-type cells. Immunocytochemical observation showed that HA-regucalcin was markedly localized in the nucleus of HA-regucalcin/ phCMV2-transfected cells. The nuclear localization was enhanced in culture with BS (5%), PTH (10(-7) M), Bay K 8644 (2.5x10(-6) M), or PMA (10(-6) M) for 24 or 48 h. Culture with staurosporine, an inhibitor of protein kinase C, caused a remarkable decrease in the localization of HA-regucalcin in the nucleus of HA-RGPR-p117/phCMV2-transfected cells with PMA. Culture with PMA (10(-6) M) for 24 or 48 h caused a remarkable increase in nuclear regucalcin protein levels. The effect of PMA in increasing nuclear regucalcin levels was completely absent in culture with staurosporine (10(-8) M). The nuclear localization of regucalcin in the stable regucalcin/pCXN2-transfected cells (transfectant) increased markedly as compared with that of wild-type cells, whereas the increase was less evident in the transfectants cultured with staurosporine. This study demonstrated that regucalcin localizes in the nucleus of cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that its nuclear localization is enhanced through an intracellular signaling process which involves protein kinase C.

Regucalcin/SMP‐30 is regulated by β‐catenin in the liver

Our laboratory has identified regucalcin as a novel target of β‐catenin in the liver. We show that regucalcin mRNA levels were downregulated in β‐catenin conditional knockout mice (KO), as compared to their wild‐type (WT) controls. Immunohistochemistry (IHC) and Western blotting (WB) confirms that regucalcin protein expression is also decreased in these mice. Analysis of HepG2 and Hep3B hepatoma cell lines demonstrates that HepG2 (active β‐catenin) have higher levels of regucalcin as compared to Hep3B (normal β‐catenin). Further, we find a correlation between the presence of β‐catenin in the nucleus or cytoplasm of human hepatocellular carcinomas (HCC) and an increase in regucalcin by IHC and WB. An analysis of the promoter region of the mouse regucalcin gene reveals at least 11 putative Tcf‐binding sites. Use of multiple deletion mutants identifies the specific sites, which might be regulating regucalcin expression by β–catenin. Finally, we examine the regucalcin expression in methionine‐choline‐deficient (MCD) diet‐induced steatohepatitis. IHC identifies an increase in regucalcin levels in MCD‐fed mice, as compared to control diet‐fed mice. In conclusion, regucalcin is a novel target of β‐catenin levels in liver and may have a biological function in liver diseases such as steatohepatitis and HCC.

Suppression of regucalcin mRNA expression in the hearts of rats administered with free radical compound: The administration-induced death is accelerated in regucalcin transgenic rats

The expression of regucalcin, a regulatory protein in the intracellular signaling system, in the hearts of rats was investigated. Regucalcin expression was examined using reverse transcription-polymerase chain reaction and Western blot analysis. Regucalcin mRNA and its protein levels in the hearts of male and female rats were significantly decreased with increasing age (50 weeks old) as compared with that of 5-week-old rats. The effect of 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a compound that produces free radical, on regucalcin mRNA expression in the hearts of female rats (5 weeks old) was examined. Heart regucalcin mRNA levels were significantly decreased at 60 or 180 min after a single intraperitoneal administration of DPPH (0.5 mg /100 g body weight), suggesting that free radical stress has a suppressive effect on the gene expression. Normal (wild) female rats died at approximately 300 min after a single intraperitoneal administration of DPPH (0.5 mg/100 g), while regucalcin transgenic (TG) female rats died at approximately 150 min after the administration. Heart regucalcin protein in DPPH-administered rats was greater in regucalcin TG rats than in normal (wild) rats. This study demonstrates that the death of regucalcin TG rats is accelerated after the administration of free radical compound, indicating that overexpression of regucalcin does not have effects as the suppressor for free radical stress and the scavenger for free radical in rats.

Overexpression of RGPR-p117 enhances regucalcin gene expression in cloned normal rat kidney proximal tubular epithelial cells

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor.

Hormonal regulation on regucalcin mRNA expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells

Regucalcin is a regulatory protein in cell signaling. This study was undertaken to determine whether regucalcin mRNA expresses in the cloned normal rat kidney proximal tubular epithelial NRK52E cells and its expression regulates due to hormones and cell signaling-related factors. Cells with subconfluency were cultured for 24, 48, or 72 h in a Dulbecco's modified Eagle medium supplemented with non-essential amino acid without bovine serum (BS). The result of Western blot analysis showed that regucalcin protein was present in the NRK52E cells. The expression of regucalcin mRNA in the cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Regucalcin mRNA expression in the NRK52E cells was significantly increased by culture with parathyroid hormone (PTH, 10(-8) or 10(-7) M), aldosterone (10(-8) or 10(-7) M), or dexamethasone (10(-8) M). The presence of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D3, 10(-8) or 10(-7) M) or calcitonin (10(-9) or 10(-8) M) did not have a significant effect on regucalcin mRNA levels in the cells. Culture with dibutyryl cyclic AMP (DcAMP, 10(-5) or 10(-4) M) or phorbol 12-myristate 13-acetate (PMA, 10(-6) M), an activator of protein kinase C, caused a significant increase in regucalcin mRNA expression. The presence of staurosporine (10(-8) M) caused a significant decrease in regucalcin mRNA expression. Dibucaine (10(-7) M), PD98059 (10(-7) M), or vanadate (10(-6) or 10(-5) M) did not have an effect on regucalcin mRNA levels. The present study demonstrates that regucalcin mRNA and its protein are expressed in the cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that the expression is enhanced by hormones which regulate ion transport in the proximal tubule.

Involvement of nuclear factor-1 (NF1) binding motif in the regucalcin gene expression of rat kidney cortex: the expression is suppressed by cisplatin administration.

The binding of nuclear factor on the promoter region of the regucalcin gene and the expression of regucalcin in the kidney cortex of rats was investigated. Nuclear extracts from kidney cortex were used for oligonucleotide competition gel mobility shift assay. An oligonucleotide between position -523 and -506 in the 5'-flanking region of the rat regucalcin gene, which contains a nuclear factor 1 (NF1) consensus motif TTGGC(N)6CC, competed with the probe for the binding of the nuclear protein from kidney cortex. The mutation of TTGGC in the consensus sequence caused an inhibition of the binding of nuclear factors. The binding of nuclear factor on the 5'-flanking region was clearly reduced in the kidney cortex obtained at 1, 2, and 3 days after a single intraperitoneal administration of cisplatin (1.0 mg/100 g body wt) to rats. Moreover, cisplatin administration caused a remarkable decrease in regucalcin mRNA levels and regucalcin concentration in the kidney cortex. Also, serum regucalcin concentration was significantly decreased by cisplatin administration. Meanwhile, serum urea nitrogen concentration was markedly elevated by cisplatin administration. The present study demonstrates that the specific nuclear factor binds to the NF1-like sequence in the promotor region of regucalcin gene in the kidney cortex of rats, and that the nuclear factor binding and regucalcin expression are suppressed by cisplatin administration.

Involvement of intracellular signaling factors in the serum-enhanced Ca2+-binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E)

The involvement of signaling factors, which are related to serum component, on the regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). H4-II-E cells were cultured for 2 or 6 h in a medium containing various reagents in the presence of serum (10% fetal bovine serum) after the subconfluent with 3-day-culture. The regucalcin mRNA expression was significantly increased by serum addition. This increase was clearly inhibited by the presence of EGTA (10(-3) M), A23187 (10(-6) M), trifluoperazine (10(-5) M), staurosporine (10(-7) M), or genistein (10(-5) M) with 6-h-culture, although the beta-actin mRNA expression was not altered by the reagents. Meanwhile, the regucalcin mRNA expression was significantly stimulated by the addition of Bay K 8644 (2.5 x 10(-6) M) in the presence of serum. This effect was also seen in the presence of genistein (10(-5) M). The present study suggests that the regucalcin mRNA expression is mediated through signaling pathways which are partly involved in Ca2+-dependent protein kinases and tyrosine kinase in H4-II-E hepatoma cells.

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (H4-II-E) is stimulated through Ca2+ signaling factors: involvement of protein kinase C.

The expression of hepatic Ca2+-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 x 10(-6) M), a Ca2+ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10(-3) M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M) or estrogen (10(-8) M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 x 10(-9) M) or dexamethasone (10(-6) M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10(-5) M), an antagonist of calmodulin, or staurosporine (10(-7) M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca2+-dependent signaling factors.

Role of calcium-binding protein regucalcin in regenerating rat liver.

Regucalcin is a novel calcium-binding protein which does not contain EF-hand motif as a Ca2+ -binding domain. The organization of the rat regucalcin gene consists of seven exons and six introns. Its mRNA is mainly present in liver but slightly in kidney with a size of 1.8 kb. Hepatic regucalcin mRNA expression is stimulated by various factors including calcium, calcitonin, insulin, and oestrogen in rats. The mRNA is also expressed in hepatoma cells (Morris hepatoma and HepG2). Regucalcin plays a role in the maintenance of cytosolic Ca2+ homeostasis in liver cells. Moreover, regucalcin has an inhibitory effect on Ca2+ /calmodulin-dependent enzyme activation, protein kinase C activation, and many Ca2+ -activated enzymes, indicating a role in the regulation of the Ca2+ -signalling system. Recently, regucalcin has been demonstrated to regulate nuclear function in liver cells. Regucalcin can inhibit Ca2+ -activated nuclear DNA fragmentation in rat isolated liver nuclei. Furthermore, the liver nuclear DNA and RNA syntheses are inhibited by regucalcin. Such an effect of regucalcin is also seen in the nuclei of regenerating rat liver. The regucalcin mRNA level is increased in regenerating liver. These findings suggest that regucalcin plays a regulatory role in the suppression for overexpression of proliferative cells.

Stimulatory effect of calcium administration on regucalcin mRNA expression is attenuated in the kidney cortex of rats ingested with saline.

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats ingested with saline was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open reading frame). Rats were freely given saline as drinking water for 7 days. Regucalcin mRNA levels in the kidney cortex were suppressed by saline ingestion. When calcium chloride (10 mg Ca/100 g body weight) was intraperitoneally administered to rats ingested with saline for 7 days, the effect of calcium administration to increase regucalcin mRNA levels was weakened by saline ingestion. Such effect was also seen by the administration of 2.5 and 5 mg Ca/100 g. Regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were not appreciably increased by the administration of calcium (10 mg/100 g). Meanwhile, calcium content in the kidney cortex was significantly elevated by the administration of calcium (10 mg/100 g) to normal rats. This increase was weakened in saline-ingested rats. Moreover, Ca2+/calmodulin-dependent protein kinase activity in the cytosol of kidney cortex was significantly decreased by saline ingestion. These results suggest the possibility that saline ingestion-induced suppression of regucalcin mRNA expression in the kidney cortex is partly involved in the attenuation of Ca2+ signalling.

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration.

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, beta-actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic beta-actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.

Tissue-specific binding of nuclear factors to the 5'-flanking region of the rat gene for calcium-binding protein regucalcin.

The existence of nuclear factors which bind to the 5'-flanking region of calcium-binding protein regucalcin gene in rats was investigated. We previously reported that rat regucalcin mRNA is expressed in a highly tissue-specific manner; the mRNA was mainly present in the liver but only slightly in the kidney. When the nuclear proteins extracted from the liver and kidney of rats were used in the gel mobility shift assays, a protein-DNA complex was uniquely formed with the DNA fragment containing the upstream region from the first exon of rat regucalcin gene. On the other hand, this complex was not found by using the nuclear extracts from rat brain, spleen, and heart. The nuclear proteins of these extracts, however, could specifically bind to the DNA fragment containing the first exon region of rat regucalcin gene, although Northern blot analysis did not show detectable amount of regucalcin mRNA levels in rat brain, spleen, and heart. The present study demonstrates that the existence of nuclear protein components which bind to the regucalcin gene. These identified components may be involved in the tissue-specific regulation of regucalcin gene expression.

Expression of calcium-binding protein regucalcin mRNA in the cloned human hepatoma cells (HepG2): stimulation by insulin.

The expression of hepatic Ca(2+)-binding protein regucalcin in the cloned human hepatoma cells (HepG2) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in HepG2 cells, although the mRNA was markedly expressed in normal rat liver. Moreover, regucalcin protein in HepG2 cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. Regucalcin mRNA expression in HepG2 cells was clearly stimulated by the culture with insulin (10(-8) M) of the effective concentration. Regucalcin protein in HepG2 cells was also increased by the treatment of insulin (10(-8) M). The present results demonstrate that regucalcin is expressed in the transformed HepG2 cells, and that the expression is stimulated by insulin.

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 protein regucalcin and its mRNA expression in mouse liver were investigated. The cDNA clone encoding a regucalcin was isolated from a mouse liver cDNA library and sequenced. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the mouse regucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouse regucalcin was composed of 299 amino acid residues, and its molecular weight was estimated to be 33,406 Da. The amino acid sequence of mouse regucalcin had 94% homology, as compared with that of rat regucalcin. Northern blot analysis with the mouse liver cDNA probe revealed that mouse regucalcin mRNA was mainly present in the liver but only slightly in the kidney with a size of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher than that of female mouse. A single intraperitoneal administration of calcium chloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced a remarkable increase in regucalcin mRNA in the liver; the increase in regucalcin mRNA levels at 30 min after calcium administration was dose-dependent. The present results demonstrate that regucalcin mRNA in mice is uniquely expressed in the liver, and that its expression is stimulated by calcium administration.

Hepatic calcium-binding protein regucalcin concentration is decreased by streptozotocin-diabetic state and ethanol ingestion in rats.

The alteration in calcium-binding protein regucalcin in the liver and serum of rats with streptozotocin (STZ)-diabetic state or ethanol ingestion was investigated. STZ (6.0 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 3 weeks later they were sacrificed by bleeding. Liver regucalcin mRNA levels were not clearly altered by the diabetic state, as evidenced by Northern blotting using regucalcin cDNA (0.9 kb of open reading frame). Based on enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG, hepatic regucalcin concentration was decreased about 50% of control levels by STZ treatment. However, serum regucalcin concentration was not significantly altered by STZ treatment. Meanwhile, when rats ingested ethanol (10 and 30%) in the drinking water for 2 weeks, liver regucalcin mRNA levels were clearly increased, although hepatic regucalcin concentration was significantly decreased. Serum regucalcin concentration was not appreciably altered. Serum transaminases (GOT and GPT) activities were significantly increased at 1 or 3 weeks after STZ administration in rats, while their activities were not altered by ethanol ingestion. The present study demonstrates that hepatic regucalcin concentration is decreased independent of mRNA expression in the STZ-diabetes and during ethanol ingestion in rats.

Calcium-binding protein regucalcin mRNA expression in the kidney cortex is suppressed by saline ingestion in rats.

The effect of adrenalectomy (ADX) or saline ingestion, which is a hypertensive factor, on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats were adrenalectomized, and 48 h later they were sacrificed. ADX caused a reduction of regucalcin mRNA levels in the kidney cortex, suggesting that adrenal glands participate in the regulation of the mRNA expression. This reduction was not restored by the subcutaneous administration of dexamethasone with an effective dose (1 mg/kg body weight), which can stimulate kidney regucalcin mRNA expression. Regucalcin mRNA levels in the kidney cortex of rats were markedly suppressed by the ingestion of saline for 7 days. The ADX-induced decrease of renal cortex regucalcin mRNA levels was not appreciably restored by saline ingestion. Moreover, regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were clearly decreased as compared with that of control (Wistar-Kyoto) rats. Meanwhile, calcium content in the kidney cortex was not significantly decreased by ADX or saline ingestion. The present study suggests that the expression of regucalcin mRNA in the kidney cortex of rats is suppressed by saline administration.

Steroid hormonal regulation of calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats.

The effect of various steroid hormones on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats received a single subcutaneous administration of steroid; the animals were sacrificed 60 min after the treatment of aldosterone (2.5, 5.0 and 10 micrograms/100 g body weight) or 6 h after the treatment of estrogen (17 beta-estradiol; 0.05, 0.1 and 0.2 mg/100 g), hydrocortisone (0.5, 1.0 and 3.0 mg/100 g) and dexamethasone (50, 100 and 150 micrograms/100 g). Regucalcin mRNA levels in the kidney cortex were clearly diminished by the administration of aldosterone or estrogen, while hydrocortisone administration had no effect. The administration of dexamethasone (100 micrograms/100 g) caused a remarkable increase of regucalcin mRNA levels in the kidney cortex. The dexamethasone-induced increase in regucalcin mRNA levels was completely blocked by the simultaneous administration of cycloheximide (150 micrograms/100 g), although the drug administration had no effect on the mRNA levels in control rats. Meanwhile, the dexamethasone administration did not cause an appreciable alteration of calcium content in the kidney cortex. The present study demonstrates that, of the various steroid hormones used, dexamethasone uniquely has a stimulatory effect on regucalcin mRNA expression in the kidney cortex of rats. The steroid effect may be mediated through a newly synthesized protein.

Enhanced expression of calcium‐binding protein regucalcin mRNA in regenerating rat liver

The expression of hepatic calcium-binding protein regucalcin mRNA was investigated in regenerating rat liver. The change of regucalcin mRNA levels was analyzed by Northern blotting, using liver regucalcin cDNA (0.9 kb with complete open reading frame). The reduced liver weight by partial hepatectomy (about 70%) was completely restored at 3 days after surgery. Regenerating liver significantly increased calcium content. Liver regucalcin mRNA levels clearly increased 1-5 days after hepatectomy, in comparison with that of sham-operated rats, although the increase was not seen 12 hr after the surgery. Increased regucalcin mRNA levels in regenerating liver were appreciably reduced by single intraperitoneal administration of actinomycin D (100 micrograms/100 g body weight), an inhibitor of transcriptional process. Moreover, the increased regucalcin mRNA levels by hepatectomy was weakened by a single intraperitoneal administration of trifluoperazine (2.5 mg/100 g), an inhibitor of Ca2+/calmodulin. These findings demonstrate that the expression of hepatic regucalcin mRNA is enhanced in regenerating rat liver.

17 beta-Estradiol stimulates the expression of hepatic calcium-binding protein regucalcin mRNA in rats.

The effect of nuclear receptor-related hormones on the expression of hepatic calcium-binding protein regucalcin mRNA in rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open-reading frame). A single subcutaneons administration of 17 beta-estradiol (0.5, 1.0 and 2.0 mg/kg body weight) in rats induced a remarkable increase of regucalcin mRNA in liver; the level was about 200% of control at 24 h after the administration of 2.0 mg/kg. The increase showed about 350% even at 6 h after the administration. Meanwhile, hepatic regucalcin mRNA level was not appreciably altered by a single subcutaneous administration of thyroxine (T4) (20, 40 and 80 mg/kg) or hydrocortisone (10 and 30 mg/kg) in rats. The present study demonstrates that the expression of hepatic regucalcin mRNA is stimulated by estrogen action in the liver nuclei of rats.

Expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats: the stimulation by calcium administration.

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex, and this expression was clearly increased by a single intraperitoneal administration of calcium chloride solution (5-15 mg Ca/100 g body weight) in rats; this increase was remarkable at 60-120 min after the administration. Thyroparathyroidectomy (TPTX) caused a slight decrease of regucalcin mRNA levels in the kidney cortex. However, the administration of calcium (10 mg/100 g) in TPTX rats produced a clear increase of regucalcin mRNA levels in the kidney cortex. The subcutaneous administration of calcitonin (10-100 MRC mU/100 g) or parathyroid hormone [1-34] (1-10 U/100 g) in TPTX rats which received calcium (10 mg/100 g) administration did not cause an appreciable alteration of regucalcin mRNA levels in the kidney cortex, suggesting that the mRNA expression is not stimulated by calcium-regulating hormones. The administration of trifluoperazine (TFP; 5 mg/100 g), an inhibitor of Ca2+/calmodulin action, completely blocked the expression of regucalcin mRNA stimulated by calcium administration. Now, calcium content in the kidney cortex was significantly elevated by a single intraperitneal administration of calcium (10 mg/100 g) in rats. The present study clearly demonstrates that the expression of regucalcin mRNA in the kidney cortex is stimulated by calcium administration in rats. This expression may be mediated through Ca2+/calmodulin action in the kidney cortex.