Satoshi Yokose

Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells.

It has been reported that PTH exerts bone-forming effects in vivo when administered intermittently. In the present study, the anabolic effects of PTH(1-34) on osteoblast differentiation were examined in vitro. Osteoblastic cells isolated from newborn rat calvaria were cyclically treated with PTH(1-34) for the first few hours of each 48-h incubation cycle. When osteoblastic cells were intermittently exposed to PTH only for the first hour of each 48-h incubation cycle and cultured for the remainder of the cycle without the hormone, osteoblast differentiation was inhibited by suppressing alkaline phosphatase activity, bone nodule formation, and mRNA expression of alkaline phosphatase, osteocalcin, and PTH/PTHrP receptor. Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and Ca2+/PKC demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, the intermittent exposure to PTH for the first 6 h of each 48-h cycle stimulated osteoblast differentiation. Both cAMP/ PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. Continuous exposure to PTH during the 48-h incubation cycle strongly inhibited osteoblast differentiation. Although both cAMP/PKA and Ca2+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. A neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin mRNA induced by the 6-h intermittent exposure. The inhibitory effect induced by the 1-h intermittent exposure was not affected by anti-IGF-I antibody. These results suggest that PTH has diverse effects on osteoblast differentiation depending on the exposure time in vitro mediated through different signal transduction systems. These in vitro findings explain at least in part the in vivo action of PTH that varies with the mode of administration.

Effects of Ionizing Radiation on Proliferation and Differentiation of Osteoblast-like Cells

Diagnostic radiation for immediate post-surgical assessment of osseointegrated dental implants has been discouraged, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. To assess this possibility, we investigated the effects of ionizing radiation on proliferation and differentiation of osteoblasts using osteoblast-like cells isolated from the calvariae of newborn rats (ROB) and a clonal osteoblastic cell line (MC3T3-E1). The cells were exposed on day 3 to a single dose of x-rays at either 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40-mGy dose from a diagnostic chest x-ray machine. The effects of radiation on cell growth and alkaline-phosphatase-specific (ALP) activity were evaluated at three-day intervals after irradiation up to day 12 in ROB cells, and evaluated at day 12 in MC3T3-E1 cells. At the culture end-point, the effects on formation of bone-like nodules were also evaluated in both ROB and MC3T3-E1 cells. Exposure of 4000 mGy differentially affected the two cell types. It inhibited cell growth and alkaline phosphatase activity in ROB cells, slightly increased alkaline phosphatase activity, and inhibited DNA content in MC3T3-E1 cells. This irradiation also strongly inhibited the formation of bone-like nodules in ROB cells. On the other hand, exposure of 40-, 100-, and 400-mGy (Linac) and 40-mGy (diagnostic quality) irradiation induced no significant changes in cell growth, alkaline phosphatase activity, and formation of bone-like nodules in ROB cells. These doses also induced no significant changes in DNA content and ALP activity in MC3T3-E1 cells. These results indicate that ionizing radiation at a single dose of up to 400 mGy induces no significant changes in cell growth and differentiation of osteoblast-like cells, at least in vitro. Higher radiation doses (4000 mGy) may exert different effects on cell proliferation and cell differentiation of osteoblasts, depending on the cell types affected. Thus, diagnostic radiation seems to have less effect on proliferation and differentiation of osteoblasts.

Age-related reduction in bone matrix protein mRNA expression in rat bone tissues: application of histomorphometry to in situ hybridization

Age-related changes in the biological activity of osteoblastic cells have been studied extensively using histomorphometry, especially in relation to osteoporosis. Nevertheless, the changes occurring in the biological activity of individual osteoblastic cells are not sufficiently clarified by this technique. In the present study, age-related changes in the expression of bone matrix protein mRNAs in individual osteoblastic cells were analyzed in vivo by in situ hybridization using undecalcified bone sections. In the femurs of 8-week-old male rats, strong expression of type I collagen and osteocalcin mRNAs was detected in cuboidal osteoblasts on the bone formation surface. Osteopontin mRNA was detected in some of the mononuclear cells and osteoclasts on the bone resorption surface, and also in osteocytes. In the femurs of 60-week-old and 100-week-old male rats, expression of these bone matrix protein mRNAs was markedly decreased. Histomorphometrical analysis of 8-week-old and 60-week-old rats indicated that both the activity and number of osteoblasts expressing type I collagen mRNA, as well as the number of osteoclasts, were reduced in these tissues in older animals. These results indicate age-related reductions in both biological activity and numbers of osteoblasts.

Cloning of rat type I receptor cDNA for bone morphogenetic protein-2 and bone morphogenetic protein-4, and the localization compared with that of the ligands

A rat homologue cDNA of mouse (Koenig et al. [1994] Mol. Cell Biol. 14:5961–5974; Suzuki et al. [1994] Proc. Natl. Acad. Sci. USA 91: 10255–10259) and human (ten Dijke et al. [1994] J. Biol. Chem. 269:16985–16988) type I receptors for BMP‐2 and BMP‐4 was cloned. Tissue distribution of the receptor mRNA was studied by in situ hybridization using rats at embryonic days 9, 13, 15, and 18 as well as 1‐ and 5‐day‐old postnatal rats. In the rats at embryonic days 9, 13, and 15, the receptor mRNA was diffusely expressed over the embryonic bodies. At embryonic day 18, the receptor mRNA expression was high in the hair and whisker follicles, tooth bud, cartilage, bone, digestive organs, lung, kidney, heart, and meninges. The receptor mRNA was expressed over a much wider area than those of the ligands in many organs. In the lung and digestive organs, the receptor mRNA was diffusely expressed and most highly expressed in the bronchial epithelium and muscle layer, respectively, in both of which mRNA expression of the ligands was undetectable. The receptor mRNA was highly expressed in the meninges, although neither of the ligands was expressed in or near this region. These results suggest that this receptor participates in both mesoderm formation in early embryogenesis and differentiation of mesodermal cells during maturation of organs, and further suggest the presence of another factor(s) that binds the type I receptor.