Expression of regucalcin in rat bone marrow cells: involvement of osteoclastic bone resorption in regucalcin transgenic rats.

The expression of regucalcin in rat bone marrow cells was investigated. The expression of regucalcin mRNA in the bone marrow cells of normal (wild-type) rat was shown by using reverse transcription-polymerase chain reaction (RT-PCR) with a specific primer of regucalcin cDNA. Regucalcin protein was detected in the marrow cells of normal (wild-type) rats using Western blot analysis. Regucalcin levels were significantly increased in the marrow cells of regucalcin transgenic (TG) male and female rats with increasing age (5-36 weeks old). When the marrow cells obtained from normal or regucalcin TG rats (36-week-old) were cultured for 7 days, the number of tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts, positive multinucleated cells (MNCs) were significantly increased in the marrow culture of regucalcin TG rats. This increase was remarkable in female TG rats as compared with male TG rats. The effect of parathyroid hormone [human PTH (1-34); 10(-7) M] or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10(-7) M] in stimulating TRACP-positive MNCs formation was significantly enhanced in female regucalcin TG rats. Calcium content in the femoral-diaphyseal and -meta-physeal tissues was significantly decreased in regucalcin TG rats (10- or 36-week-old). This decrease was greater in female than in male. Femoral-metaphyseal deoxyribonucleic acid (DNA) content was significantly reduced in regucalcin TG male and female rats (36-week-old). Moreover, serum inorganic phosphorus, triglyceride, HDL-cholesterol, and albumin concentrations were significantly increased in regucalcin TG female rats (36-week-old), while serum calcium, zinc, and glucose concentrations were not significantly altered in TG male and female rats. In TG male rats, serum triglyceride and HDL-cholesterol concentrations were significantly raised. This study demonstrates that regucalcin is expressed in rat bone marrow cells, and that osteoclastic bone resorption is stimulated in regucalcin TG rats with increasing age. Also, regucalcin TG aged rats was found to induce serum metabolic disorder.

Enhancement of albumin expression in bone tissues with healing rat fractures.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

Novel protein RGPR-p117: the gene expression in physiologic state and the binding activity to regucalcin gene promoter region in rat liver.

The binding activity of a novel regucalcin gene promoter region-related protein (RGPR-p117) to the TTGGC sequence of the rat regucalcin gene promoter region was investigated. The expression of RGPR-p117 mRNA was seen in the liver tissues of male and female rats. The sexual difference of this expression was not found. Liver RGPR-p117 mRNA expression was not changed with increasing age (1-50 weeks old), and its expression was not altered by fasting or refeeding. Nuclear factor I-A1 (NF1-A1) has been identified to be a transcription factor in stimulating the rat regucalcin gene promoter activity (Misawa and Yamaguchi [2002a] J Cell Biochem 84:795-802]. Recombinant nuclear factor I-A1 (NF1-A1) and RGPR-p117 proteins were used gel mobility shift assay. RGPR-p117 could not bind to TTGGC motif of the sequence between -525 and -504, which has been defined as a functional promoter element II-b. NF1-A1 was specifically bound to the II-b oligonucleotide. Moreover, RGPR-p117 was not bound to the II-b oligonucleotide in the presence of NF1-A1 or rat liver nuclear protein. The binding of NF1-A1 to the II-b oligonucleotide was not altered in the presence of RGPR-p117. This study demonstrates that RGPR-p117 mRNA, is expressed stably for physiologic change in rat liver, and that recombinant the protein does not directly bind to the TTGGC motif in rat regucalcin gene promoter.

Identification of bioactive molecules by adipogenesis profiling of organic compounds.

An important step in the postgenomic drug discovery is the construction of high quality chemical libraries that generate bioactive molecules at high rates. Here we report a cell-based approach to composing a focused library of biologically active compounds. A collection of bioactive non-cytotoxic chemicals was identified from a divergent library through the effects on the insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic and sensitive morphological alterations in cultured mammalian cells. The resulting focused library amply contained unique compounds with a broad range of pharmacological effects, including glucose-uptake enhancement, cytokine inhibition, osteogenesis stimulation, and selective suppression of cancer cells. Adipogenesis profiling of organic compounds generates a focused chemical library for multiple biological effects that are seemingly unrelated to adipogenesis, just as genetic screens with the morphology of fly eyes identify oncogenes and neurodegenerative genes.

Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats.

The potential sensitivity of liver specific protein regucalcin as a biochemical marker of chronic liver injury with carbon tetrachloride (CCl4) administration in rats was investigated. CCl4 (10%; 1.0 ml/100 g body wt) was orally given 5 times at 3-day intervals to rats, and the animals were killed by bleeding at 3, 6, 18, and 30 days after the first administration of CCl4. The body weight of rats was significantly lowered 3 and 6 days after CCI4 administration as compared with that of control rats administered with corn oil, and then the weight was restored at 18 and 30 days. Serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities were significantly increased 3 days after the administration, while a significant increase in serum y-glutamyltranspeptidase (gamma-GTP) activity was seen at 3 and 6 days after the administration. Serum GOT, GPT, and gamma-GTP activities were restored to control levels at 18 and 30 days after CCl4 administration. Serum albumin, alpha-fetoprotein, and ammonium levels were not changed by CCl4 administration. Meanwhile, serum regucalcin concentration was markedly increased 3 and 6 days after CCl4 administration, and a significant increase in serum regucalcin concentration was observed 18 and 30 days after the administration. Liver regucalcin mRNA and liver cytosolic regucalcin levels were significantly decreased 18 and 30 days after CCl4 administration. Liver content of calcium, which intracellular calcium homeostasis is maintained, was significantly increased between 3 and 30 days after CCl4 administration. Hepatic mitochondrial succinate dehydrogenase activity was significantly increased 30 days after the administration. The present study demonstrates that serum regucalcin has a potential sensitivity as a specific biochemical marker of chronic liver injury with CCl4 administration in rats.

Role of endogenous regucalcin in bone metabolism: bone loss is induced in regucalcin transgenic rats.

The role of endogenous regucalcin in the regulation of bone metabolism was investigated by using regucalcin transgenic (TG) rats. The expression of regucalcin mRNA in the femoral-diaphyseal and -metaphyseal tissues of normal (wild-type) rats was shown by using reverse transcription-polymerase chain reaction (RT-PCR) with a specific primer of regucalcin cDNA. Regucalcin protein was detected in the femoral-diaphyseal and -metaphyseal tissues of normal (wild-type) rats using Western analysis. Regucalcin levels were significantly increased in the femoral-metaphyseal tissues of regucalcin TG male rats and in the diaphyseal and metaphyseal tissues of the TG female rats. The morphologic change in the femoral-diaphyseal and -metaphyseal tissues of regucalcin TG rats was demonstrated by using a peripheral quantitative computed tomography (pQCT); morphologic change was great in the femoral tissues of female rats as compared with that of male rats. Mineral content, mineral density and polar strength strain index in the femoral-diaphyseal and -metaphyseal tissues were markedly reduced in regucalcin TG female rats. A significant decrease in cortical thickness was seen in the femoral diaphysis of regucalcin TG female rats. Calcium content in the femoral-diaphyseal and -metaphyseal tissues was significantly decreased in regucalcin TG male and female rats; a remarkable decrease was seen in female rats. Femoral-metaphyseal alkaline phosphatase activity was significantly lowered in regucalcin TG female rats. The enzyme activity was not significantly changed in the femoral-diaphyseal tissues of the TG female rats. In the diaphyseal tissue of male rats, the enzyme activity was significantly decreased in the TG rats. A significant decrease in deoxyribonucleic acid (DNA) content was seen in the metaphyseal tissue of regucalcin TG male rats and in the diaphyseal and metaphyseal tissues of the TG female rats. This study demonstrates that bone loss is induced in the femoral tissue of regucalcin transgenic rats, and that a remarkable decrease in bone morphologic index and biochemical component was seen in the female rats. Regucalcin may be involved in the regulation of bone metabolism.

Gene expression for a novel protein RGPR-p117 in various species: the stimulation by intracellular signaling factors.

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors.

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.

Identification of transcription factor in the promoter region of rat regucalcin gene: binding of nuclear factor I-A1 to TTGGC motif.

Hepatic nuclear protein has been reported to bind specifically to the TTGGC sequence of the rat regucalcin gene promoter region in stimulating the promoter activity (Misawa and Yamaguchi [2000] Biochem. Biophys. Res. Commun. 279: 275-281). The present study was undertaken to identify transcription factor, which binds to TTGGC motif in the rat regucalcin gene promoter, using the yeast one-hybrid system. The sequence between -525 and -504, which has been defined as a functional promoter element II-b, was used as bait to screen a rat liver cDNA library. Two cDNA clones were identified as a nuclear factor I-A1 (NF1-A1). The results of gel mobility shift assay and mutation analysis using recombinant NF1-A1 protein showed that this protein could specifically bind to TTGGC motif of the II-b oligonucleotide in promoter region. The expression of NF1-A1 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. This study demonstrates that NF1-A1 is a transcription factor in stimulating the rat regucalcin gene promoter activity.