Yoshihiro Miwa

Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3‐E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18‐ and 24‐mer antisense oligonucleotides caused concentration‐dependent increases in the number of mineralized nodules, acid‐soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide‐treated MC3T3‐E1 cells, thickened extracellular matrix, well‐developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3‐E1 cells. Furthermore, MC3T3‐E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose‐dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322–332, 2006.

cDNA cloning of bovine thrombospondin 1 and its expression in odontoblasts and predentin

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Putrescine-stimulated intracellular Ca2+ release for invasiveness of rat ascites hepatoma cells

Our previous study showed that treatment of highly invasive rat ascites hepatoma (LC‐AH) cells with α‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, decreased both their intracellular level of putrescine and their in vitro invasion of a monolayer of calf pulmonary arterial endothelial (CPAE) cells, and that both these decreases were completely reversed by exogenous putrescine, but not spermidine or spermine. Here we show that all adhering control (DFMO‐untreated) cells migrated beneath CPAE monolayer with morphological change from round to cauliflower‐shaped cells (migratory cells). DFMO treatment increased the number of cells that remained round without migration (nonmigratory cells). Exogenous putrescine, but not spermidine or spermine, induced transformation of all nonmigratory cells to migratory cells with a concomitant increase in their intracellular Ca2+ level, [Ca2+]i. The putrescine‐induced increase in their [Ca2+]i preceded their transformation and these effects of putrescine were not affected by antagonists of the voltage‐gated Ca2+ channel, but were completely suppressed by ryanodine, which also suppressed the invasiveness of the control cells. The DFMO‐induced decreases in both [Ca2+]i and the invasiveness of the cells were restored by thapsigargin, which elevated [Ca2+]i by inhibiting endoplasmic Ca2+‐ATPase, indicating that thapsigargin mimics the effects of putrescine. These results support the idea that putrescine is a cofactor for Ca2+ release through the Ca2+ channel in the endoplasmic reticulum that is inhibited by ryanodine, this release being initiated by cell adhesion and being a prerequisite for tumor cell invasion.

A MUTANT STRAIN (LEC) OF RAT WITH LOW DEGREE OF ZINC-INDUCED HEPATIC METALLOTHIONEIN GENE EXPRESSION

A mutant strain (LEC) of rats was found to possess the feature of low degree of the zinc-induced hepatic metallothionein (MT) gene expression due to an alteration of the transcription factor concerned in the gene expression. Northern blot analyses showed that the amount of MT-1 mRNA induced by intraperitoneal zinc injection is smaller in LEC mutant rat liver than in normal rat liver, while the amount of MT-1 mRNA induced by copper injection is indistinguishable between LEC and normal rat livers. Gel retardation assays showed that LEC and normal rat livers are different in the nuclear protein which binds to the metal-responsive element (MRE) of the MT gene in a zinc-dependent manner, and that the efficiency of the zinc-dependent binding of the nuclear protein to the MRE is lower in LEC rat liver than in normal rat liver. LEC rat should provide a useful model to understand the transcription factor concerned in the MT gene expression by zinc.