Akemi Inaba

Serum Calcium-decreasing Factor, Caldecrin, Inhibits Osteoclast Differentiation by Suppression of NFATc1 Activity

Caldecrin/chymotrypsin C is a novel secretory-type serine protease that was originally isolated as a serum calcium-decreasing factor from the pancreas. Previously, we reported that caldecrin suppressed the bone-resorbing activity of rabbit mature osteoclasts (Tomomura, A., Yamada, H., Fujimoto, K., Inaba, A., and Katoh, S. (2001) FEBS Lett. 508, 454–458). Here, we investigated the effects of caldecrin on mouse osteoclast differentiation induced by macrophage-colony stimulating factor and the receptor activator of NF-κB ligand (RANKL) from the monocyte/macrophage cell lineage of bone marrow cells. Wild-type and protease-deficient mutant caldecrin dose-dependently inhibited RANKL-stimulated tartrate-resistant acid phosphatase-positive osteoclast formation from bone marrow cells. Caldecrin did not affect macrophage colony formation from monocyte/macrophage lineage cells or osteoclast progenitor generation in cultures of bone marrow cells. Caldecrin inhibited accumulation of the RANKL-stimulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) mRNA in bone marrow cells, which is a key transcription factor for the differentiation of osteoclasts. Caldecrin also suppressed RANKL-induced differentiation of the RAW264.7 monocyte/macrophage cell line into osteoclasts. Caldecrin reduced the transcriptional activity of NFATc1 in RAW264.7 cells, whereas those of NF-κB and c-Fos, which are also transcription factors involved in osteoclast differentiation, were unaffected. Caldecrin inhibited RANKL-stimulated nuclear translocation of NFATc1 and the activity of the calcium/calmodulin-dependent phosphatase, calcineurin. Caldecrin inhibited phospholipase Cγ1-mediated Ca2+ oscillation evoked by RANKL stimulation. RANKL-stimulated phosphorylation of spleen tyrosine kinase (Syk) was also attenuated by caldecrin. Taken together, these results indicate that caldecrin inhibits osteoclastogenesis, without its protease activity, by preventing a phospholipase Cγ1-mediated Ca2+oscillation-calcineurin-NFATc1 pathway.

Role of the conserved Ser–Tyr–Lys triad of the SDR family in sepiapterin reductase

Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser-Tyr-Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.

Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin).

We previously reported on the serum calcium‐decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315–30321]. To address the mechanism of this serum calcium‐decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone‐resorbing activity. Wild‐type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose‐dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease‐deficient mutant caldecrins. Recombinant procaldecrin (−13–239), and its fragments (−13–125), (1–111), (1–46), (47–111), and (126–239) were expressed as His‐tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (−13–239) and fragment (−13–125) did not affect the suppressive activity, whereas fragments (1–111) and (126–239) did suppress the bone resorption. The bone‐resorbing activity was also suppressed by fragment (47–111), not by fragment (1–46). Overlapping fragments (47–62), (47–79), (47–98), (56–111), (71–111), and (85–111) were compared for their suppressive activity. The fragments (47–62) and (85–111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71–79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71–79) is responsible for the suppression of bone resorption by caldecrin.

GENETIC DIFFERENTIATION IN JAPANESE FRESHWATER CRAB, GEOTHELPHUSA DEHAANI (WHITE) : ISOZYME VARIATION AMONG NATURAL POPULATIONS IN KANAGAWA PREFECTURE AND TOKYO

Abstract In Kanagawa Prefecture and its surrounding area, two types of body color populations (DA: dark and BL: blue) of Japanese freshwater crab, Geothelphusa dehaani, are distributed parapatrically, suggesting some extent of genetic differentiation between them. In this study we examined the genetic variation of G. dehaani in Kanagawa Prefecture and Tokyo by means of electrophoretic analysis on 5 enzyme loci. The results clearly show the existence of substantial genetic differentiation among different color type populations. In addition, some extent of differentiation is also observed within the BL populations.

Transient Activation of Dihydropteridine Reductase by Ca2+-activated Proteolysis

Abstract A transient activation of dihydropteridine reductase (DHPR), which is the regenerating enzyme of tetrahydrobiopterin in the system of aromatic amino acid hydroxylases, was studied during the incubation of DHPR with Ca2+-activated protease, m-calpain. The DHPR subunit (29 k) was cleaved by m-calpain just before the 35th (Ser) and 48th (Val) residue from the N-terminus, generating two new fragments of 21 k and 19 k. By determining kinetic parameters, we found that 21 k and 19 k were more active than the native enzyme and that the activation of them was more remarkable and transient against the natural substrate of quinonoid dihydrobiopterin than against a synthesized substrate. Phosphorylation of DHPR by Ca2+/calmodulin-dependent protein kinase II controlled the sensitivity of the enzyme to the Ca2+-activated protease.

Determination by Site-directed Mutagenesis of Sites in Sepiapterin Reductase Phosphorylated by Ca2+/Calmodulin-dependent Protein Kinase II

Summary Phosphorylation sites of sepiapterin reductase (SPR) phosphorylated by Ca2+-dependent protein kinase II (CaM KII) were studied. By immunoreaction against phosphorylated amino acids, we found that Ser residues of SPR were phosphorylated. We constructed several point mutants of SPR by site-directed mutagenesis and expressed then in E. coli. In assays with anti-phospho Ser antibody, we determined that each of the three Ser residues, S46, S 196, and S214, of SPR was phosphorylated by CaM KIl. Each of these serine residues in SPR was found in a CaM KII phosphorylation site sequelce (Arg-X-X-Ser/Thr).