Noriaki Shimokawa

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The cDNA of a Ca2+‐binding protein regucalcin was cloned from a rat liver cDNA library which was constructed in λZAPII by immunoscreening. Positive clones were obtained from which spanned the region of interest, and they gave a sequence of 1.7 kb by sequencing with the dideoxynucleotide method. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the regucalcin molecule. Regucalcin was composed of 299 amino acid residues and its molecular weight was estimated to be 33,388 Da. The hydropathy profile of regucalcin showed a highly hydrophilic character. The nucleotide and amino acid sequences of regucalcin did not have statistically significant homology, as compared with the registered sequences which are found in the EMBL and GenBank databases containing several other Ca2+‐binding proteins (calmodulin, calbindin‐D28k and S‐100β). The regucalcin molecule did not contain the EF‐hand motif as a Ca2+‐binding domain. The present study demonstrates that regucalcin is a unique Ca2+‐binding protein in the liver of rats.

Calcium administration stimulates the expression of calcium-binding protein regucalcin mRNA in rat liver

The distribution and expression of mRNA encoding the Ca2+‐binding protein regucalcin in rats were investigated by Northern blot analyses. Liver regucalcin cDNA (0.6 kb) was used as a probe. The analyses of total RNAs extracted from various tissues of rat indicated that regucalcin mRNA was mainly present in liver but only slightly in kidney with a size of 1.8 kb. The expression level decreased with increasing age (3, 10 and 25 weeks). A single intraperitoneal administration of calcium chloride (15 mg Ca/100 g body weight) induced a remarkable increase in regucalcin mRNA in liver; the level was about 200% of control at 30 min after the administration. Subsequently, the expression level began to decrease with time and was about 40% of control level at 120 min after the administration. The increase in regucalcin mRNA levels at 30 min after calcium administration was dose‐dependent. These observations show that the expression of regucalcin mRNA is specific in liver of various tissues, and that it is regulated by Ca2+ administration. Regucalcin may have a role as regulatory protein for calcium homeostasis in liver cells.

Genomic cloning and chromosomal assignment of rat regucalcin gene

The gene for a Ca2+-binding protein regucalcin was cloned from a rat genomic library which was constructed in λ FIX II by screening with radiolabeled probe (complementary DNA of rat liver regucalcin). Positive clone had 19.9 kb insert of size and contained four exons of the gene coding for a rat regucalcin. These exons included the partial coding sequence (61.2% of open reading frame) and the entire 3′-untranslated region of the gene. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. The sequence analysis of the clone showed that the identifier sequence and two simple repeated sequences exist in the intron of the gene. Moreover, chromosomal location of the rat regucalcin gene was determined by direct R-banding fluorescencein situ hybridization (FISH) method with the 19.9 kb clone containing four exons. The regucalcin gene was localized on rat chromosome Xq11.1–12 proximal end.

Expression of hepatic calcium-binding protein regucalcin mRNA is mediated through Ca2+/calmodulin in rat liver.

The effect of signal transduction‐related factors was investigated to clarify the expression mechanism for mRNA of the hepatic Ca2+‐binding protein regucalcin in the liver of rats. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.6 kb). A single intraperitoneal administration of calcium chloride (15 mg Ca2+: 0.374 mmol/100 g body weight) to rats induced a remarkable increase of regucalcin mRNA in liver; the level was about 170% of controls at 30 min after administration. This increase was completely inhibited by simultaneous administration of trifluoperazine (5.0 mg/100 g), an antagonist of calmodulin. On the other hand, a single intraperitoneal administration of phorbol ester or dibutyryl cAMP (10–1,000 μg/100 g) did not cause a significant alteration of hepatic regucalcin mRNA levels. Also, administration of zinc, copper and cadmium (0.374 mmol of metal ion/100g) did not have an appreciable effect on hepatic regucalcin mRNA levels. These findings demonstrate that the expression of hepatic regucalcin mRNA is mediated through Ca2+/calmodulin.

The 5′ end sequences and exon organization in rat regucalcin gene

The 5−flanking region of the gene for a Ca2−-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda EMBL3 SP6/T7 vector. The genomic library was screened by using the radiolabeled probe with the 5′ region (0.5 kb) of rat regucalcin complementary deoxyribonucleic acid (cDNA). Positive clone had the 5.5 kb fragment which was hybridized with the 5′-probe. This fragment contained three exons (I–III) of the gene coding for a rat regucalcin. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. A supposed translational initiation site existed in the exon 11. Homology analysis showed that a putative transcription start site in the rat regucalcin gene was located at position 26 downstream from a TATA-box. Another upstream element, a CCAAT box-like sequence, was located at −170. Moreover, there were many regulatory elements (Hox, AP-1, AP-2 and AP-4) in the 5′-flanking region of the rat regucalcin gene. The organization of rat regucalcin gene seemed to be about 18 kb in size and consisted of seven exons and six introns.

Hepatic calcium-binding protein regucalcin is released into the serum of rats administered orally carbon tetrachloride

The change in calcium-binding protein regucalcin, mainly localized in liver, in the liver and serum of rats received a single oral administration of carbon tetrachloride (50%; 1.0 ml/100 g body weight) was investigated. The change of regucalcin mRNA levels in the liver was analyzed by Northern blotting using liver regucalcin cDNA (0.6 kb). At 10 and 24 h after the administration, liver regucalcin mRNA levels were reduced markedly. Moreover, regucalcin concentration in the liver and serum was estimated by enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG. Administration of carbon tetrachloride (CCl4) induced a significant decrease in liver regucalcin concentration and a corresponding elevation of serum regucalcin concentration at 24 h after the administration. An appreciable increase in serum regucalcin concentration was seen at 2 h after the administration. Meanwhile, serum transaminases (GOT and GPT) activities were significantly increased by CCl4 administration, indicating that liver injury is induced. The present study demonstrates that hepatic regucalcin is released into the serum of rats administered orally CCl4, suggesting that the estimation of serum regucalcin is a useful tool for diagnosis of liver injury.

Specific species and tissue differences for the gene expression of calcium-binding protein regucalcin

The existence and expression of gene encoding the Ca2+-binding protein regucalcin in various species and tissues were investigated with Southern and Northern hybridization analyses using regucalcin cDNA (0.9 kb of open reading frame). Genomic Southern hybridization analysis demonstrated that regucalcin gene was widely conserved among higher animals including human, monkey, rat, mouse, dog, bovine, rabbit and chicken. The gene was not found in yeast. The Northern blot analysis of poly (A)+RNAs extracted from the liver of various species showed that regucalcin mRNA was predominantly expressed in rat and mouse, although the expression was also seen in human, bovine and chicken. Furthermore, the enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG indicated that hepatic regucalcin concentration was most pronounced in rat as compared with that of guinea pig, mouse and chicken. These observations show that the gene expression of regucalcin and its protein synthesis is unique in the liver of rats, suggesting the existence of a specific mechanism in demonstrating regucalcin synthesis from gene.

Inhibitory Effect of β-Alanyl-L-Histidinato Zinc on Bone Resorption in Tissue Culture

The inhibitory effect of beta-alanyl-L-histidinato zinc (AHZ) on bone resorption in tissue culture was investigated. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 48 h in Dulbeccos modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-4) mol/l AHZ. The bone-resorbing factors, parathyroid hormone (1-34) (PTH; 10(-7) mol/l), prostaglandin E2 (10(-5) mol/l), interleukin-1 alpha (IL1 alpha; 50 U/ml), and lipopolysaccharide (10 micrograms/ml), caused a significant decrease in bone calcium content. The decreases in bone calcium content induced by bone-resorbing factors were completely inhibited by the coexistence of AHZ (10(-6) to 10(-4) mol/l). Also, AHZ (10(-5) mol/l) completely inhibited the PTH (10(-7) mol/l) or IL1 alpha (50 U/ml)-induced increase in medium glucose consumption and lactic acid production by bone tissue. Furthermore, AHZ (10(-5) mol/l) fairly blocked both PTH (10(-7) mol/l)-increased acid phosphatase and decreased alkaline phosphatase activities of bone tissue. The inhibitory effect of AHZ (10(-5) mol/l) on PTH (10(-7) mol/l)-stimulated bone resorption was clearly prevented by the presence of 10(-4) mol/l dipicolinate, a chelator of zinc. However, zinc sulfate (10(-7) to 10(-4) mol/l) did not inhibit the PTH (10(-7) mol/l)-stimulated bone resorption in tissue culture. These findings indicate that AHZ had a direct inhibitory effect on bone resorption in vitro, and the AHZ effect was found in the chemical form of zinc-chelated dipeptide.

Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: the hormonal effect is mediated through calcium.

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca2+-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25–100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca2+ in rat liver.

Expression of hepatic calcium-binding protein regucalcin mRNA is decreased by phenobarbital administration in rats.

The effect of phenobarbital on the expression of calcium-binding protein regucalcin mRNA in rat liver was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open reading frame). Phenobarbital (4, 8 and 12 mg/ 100 g body weight) was intraperitoneally administered to rats 3 times with 24 h intervals, and the animals were sacrificed by bleeding at 24 h after the last administration. The hepatic regucalcin mRNA levels were markedly reduced by phenobarbital administration. This decrease was about 50% of control level with the 12 mg/100 g dose. Moreover, the hepatic regucalcin concentration was significantly decreased by the administration of phenobarbital (12 mg/100 g), although the serum regucalcin concentration was not altered appreciably. Meanwhile, serum transaminases (GOT and GPT) activities were not increased by the administration of phenobarbital (4 and 12 mg/100 g). The present study demonstrates that the expression of hepatic regucalcin mRNA is decreased by phenobarbital administration in rats, suggesting that regucalcin does not have a role in drug metabolism related to phenobarbital.