Osamu Nakade

MELATONIN STIMULATES PROLIFERATION AND TYPE I COLLAGEN SYNTHESIS IN HUMAN BONE CELLS IN VITRO

Abstract: The pineal secretory product melatonin reportedly regulates release of growth hormone in humans and prevents phototherapy‐induced hypocalcemia in newborn rats, suggesting that melatonin affects bone metabolism. Little is known about the effects of melatonin on bone in vitro or in vivo. The present study was undertaken to examine whether melatonin acts directly on normal human bone cells (HOB‐M cells) and human osteoblastic cell line (SV‐HFO cells) to affect osteogenic action in vitro. The effect of melatonin on bone cell proliferation was determined using the 2, 3‐bis[2‐methoxy‐4‐nitro‐5‐sulfophenyl]‐2H‐tetrazolium‐5‐carboxanilide (XTT) assay after a 24 hr incubation with melatonin. Melatonin significantly and dose‐dependently increased the proliferation in HOB‐M cells and SV‐HFO cells by 215 ± 22.1%, and 193 ± 6.4%, respectively, with a maximal effect at a concentration of 50 μM. To evaluate the effect of melatonin on bone cell differentiation, alkaline phosphatase (ALP) activity, osteocalcin secretion and procollagen type I c‐peptide (PICP) production (a measure of type I collagen synthesis) were measured after a 48 hr treatment. While melatonin at micromolar concentrations did not significantly affect either the ALP activity or the osteocalcin secretion, it significantly and dose‐dependently increased the PICP production in HOB‐M cells and SV‐HFO cells by 983 ± 42.2%, and 139 ± 4.2%, respectively, with the maximal stimulatory doses between 50 and 100 μM. These results provide new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells in vitro, suggesting that melatonin may act to stimulate bone formation.

Melatonin at Pharmacologic Doses Increases Bone Mass by Suppressing Resorption Through Down-Regulation of the RANKL-Mediated Osteoclast Formation and Activation†

This study evaluated if melatonin would increase bone mass in mice. Four groups of 4‐week‐old male ddy mice received daily injections of vehicle or 1, 5, or 50 mg/kg of melatonin, respectively, for 4 weeks. Treatment with 5 mg/kg per day or 50 mg/kg per day of melatonin significantly increased bone mineral density (BMD; by 36%, p < 0.005) and bone mass (bone volume per tissue volume [BV/TV] by 49%, p < 0.01, and trabecular thickness [Tb.Th] by 19%, p < 0.05). This treatment significantly reduced bone resorption parameters (i.e., osteoclast surface [Oc.S/bone surface {BS}] by 74%, p < 0.05, and osteoclast number [N.Oc/BS] by 76%, p < 0.005) but did not increase histomorphometric bone formation parameters (i.e., bone formation rate [BFR/BS], mineral apposition rate [MAR], and osteoid volume [OV/TV]), indicating that melatonin increases bone mass predominantly through suppression of bone resorption. Melatonin (1–500 μM) in vitro caused dose‐dependent reduction (p < 0.001 for each) in the number and area of resorption pits formed by osteoclasts derived from bone marrow cells but not those formed by isolated rabbit osteoclasts. Because RANKL increases, while osteoprotegerin (OPG) serves as a soluble decoy receptor for RANKL to inhibit osteoclast formation and activity, the effect of melatonin on the expression of RANKL and OPG in mouse MC3T3‐E1 osteoblastic cells was investigated. Melatonin (5–500 μM) increased in a dose‐dependent manner and reduced the mRNA level of RANKL and both mRNA and protein levels of OPG in MC3T3‐E1 cells (p < 0.001 for each). In summary, these findings indicated for the first time that melatonin at pharmacologic doses in mice causes an inhibition of bone resorption and an increase in bone mass. These skeletal effects probably were caused by the melatonin‐mediated down‐regulation of the RANKL‐mediated osteoclast formation and activation.

Effect of extracellular calcium on the gene expression of bone morphogenetic protein-2 and -4 of normal human bone cells

Abstract A high extracellular calcium level inhibits the formation of osteoclast-like cells and stimulates osteoblastic proliferation, indicating that extracellular calcium plays an important role in the process of bone remodeling. The present study examined the effects of a high extracellular calcium level on mRNA levels of bone morphogenetic protein (BMP)-2 and -4, which are well-documented osteoinductive proteins, and the differentiation of normal human mandible-derived bone cells in vitro. High extracellular calcium significantly increased cell proliferation at an optimal dose of 0.4 mM CaCl2 added to control medium containing 1.8 mM CaCl2. The addition of 0.1–0.4 mM CaCl2 markedly increased the mRNA levels of BMP-2 and -4 following incubation for 0.5 and 24 h as evaluated by reverse transcription–polymerase chain reaction. While an increased extracellular calcium level (addition of 0.1–1.2 mM CaCl2) failed to increase alkaline phosphatase activity and osteocalcin secretion, it did significantly increase type I collagen synthesis, monitored by the production of procollagen type I carboxy-terminal peptide. These results indicate that the extracellular calcium level regulates BMPs and type I collagen synthesis in osteoblastic cells.

Stimulation by low concentrations of fluoride of the proliferation and alkaline phosphatase activity of human dental pulp cells in vitro

Fluoride has been used for decades, either systemically or topically, to prevent dental caries. The purpose of this study was to clarify the effects of low concentrations of fluoride on proliferation, differentiation and extracellular-matrix synthesis in normal human dental pulp cells (DP-1 and DP-2) in vitro. The effects were compared with those on a human osteoblastic osteosarcoma cell line, TE-85. Fluoride at micromolar concentrations significantly and dose-dependently stimulated [3H]thymidine incorporation into DNA in DP-1, DP-2 and TE-85 cells, with optimal effects around 50 microM, by 127 +/- 7%, 124 +/- 0.6% and 152 +/- 13.4%, respectively. To assess the potential influence of fluoride on cell differentiation, the effects of mitogenic concentrations on alkaline phosphatase activity were measured. Fluoride significantly increased the enzymes activity in DP-1 and TE-85 by 177 +/- 12% and 144 +/- 12.3%. To evaluate the effect on extracellular-matrix synthesis, the synthesis of type I collagen was indirectly determined by an assay of procollagen type I c-peptide production. Fluoride significantly increased that production by 150 +/- 8.7% in TE-85, but not in either DP-1 or DP-2. These observations suggest that fluoride, if used at low concentrations, could be a useful therapeutic agent where increased regeneration of dentine is desired, such as after pulp amputation, by stimulating the proliferation and differentiation of the dental pulp cells.

Evidence for the involvement of bone morphogenetic protein-2 in phenytoin-stimulated osteocalcin secretion in human bone cells.

Recent work has shown that the actions of phenytoin on bone cell proliferation and differentiation are, in part, mediated through the upregulation of transforming growth factor-beta1 (TGF-beta(1)). The present study was undertaken to examine the effect of phenytoin on bone morphogenetic proteins (BMP)-2 and -4, which are well-recognized osteoinductive proteins of the TGF-beta superfamily, in osteoblastic cells. Treatment with 5-50 microM of phenytoin increased the amount of mRNA for BMP-2 after a 0.5-24 h incubation in normal human mandible-derived bone cells (HOB-M cells), but failed to affect the mRNA for BMP-4. Phenytoin treatment for 48 h significantly increased the secretion of BMP-2 by approx. four-fold, at an optimal concentration of 10 microM. While TGF-beta(1) inhibited osteocalcin secretion from HOB-M cells, both phenytoin and BMP-2 significantly stimulated it. Importantly, the stimulatory effects of phenytoin on osteocalcin release were completely blocked by the neutralizing antihuman BMP-2 monoclonal antibody. These results indicate that the stimulatory action of phenytoin on osteocalcin secretion in normal human bone cells is mediated, at least partly, through the upregulation of BMP-2, rather than that of TGF-beta(1).

Alpha1-antitrypsin deficiency and toxic shock: a Japanese autopsy case.

A 74‐year‐old Japanese female presented with the sudden appearance of hemorrhagic purpuric ecchymoses on her lower extremities and with fever and chills, and died on the fifth day of hospitalization. A diagnosis of α1‐antitrypsin (AT) deficiency was made postmortem. The liver weighed 1260 g. Histological sections from the liver revealed rather severe fatty changes of the hepatocytic parenchyma and partial loss of the normal hepatic architecture with fibrosis. The hepatocytes contained periodic acid‐Schiff (PAS)‐positive, diastase‐resistant and α1‐AT‐positive intracytoplasmic globules. There was markedly increased inflammatory infiltration with severe edema and congestion, accompanied by fibrous, thickened pulmonary alveolar walls with fibrin deposition in the lungs (right, 410 g; left, 280 g), which suggest findings similar to those seen in multiple organ failure. Mild pulmonary emphysema was also present in the upper lobes of the lungs. Histological sections from the hemorrhagic necrotic ecchymoses of the skin showed marked neutrophil infiltration over the subcutaneous tissue with bleeding and blistering. A finding of thrombophlebitis was also found in the subcutaneous tissue. No bacteria were detected in the ecchymoses, the urine or the blood. Plasma protein analysis revealed a lower level (9.5 μmol/L) of α1‐AT and a higher level (330 U) of anti‐streptolysin O (ASO). These findings suggest that the patient died of toxic shock‐like syndrome and that α1‐AT deficiency might have facilitated the development of the toxic shock. To our knowledge, this is the first case of toxic shock associated with α1‐AT deficiency.

Immunohistochemical study on carcinogenesis of the oral mucosa of the hamster. I. Localization of keratin in carcinomas of cheek pouch mucosa.

The distribution of intracellular keratin was studied in dysplasia and squamous cell carcinomas of the cheek pouch mucosa of the hamster induced by the application of a 0.5% DMBA mineral oil. The hamster cheek pouchs were excised, and placed in a cold aqueous solution of 1% acetic acid for 1 hour while stirring. The pouches were rinsed and placed in a shallow pan of distilled water. The epithelium was then gently freed from the underlying connective tissue with the use of wooden chopsticks. With this technique the epithelium was easily separated from the connective tissue. anti-keratin antibody was induced in rabbits against total keratin extracts from the squamous epithelium of the cheek pouch mucosa. The serum was tested by the indirect immunoperoxidase method (PAP method). Chilled acetonefixed, paraffin-embedded tissues were employed.Anti-keratin antibody against total keratin was directed toward cytoplasmic antigen of keratinocytes of spinous and granular layers of the hamster cheek pouch epithelium. No labeling could be detected in the basal cell layer. In the dysplastic lesions and squamous cell carcinomas the expression of keratin antigens was markedly diminished or not detected.The present study suggests that the difference of the staining intensities of keratin among the normal epithelium, dysplasia and squamous cell carcinoma is a valuable indicator of the malignant transformation in the cheek pouch mucosa.