Tohru Kaku

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.

Role of β-defensins in oral epithelial health and disease

The oral epithelium functions as a mechanical and protective barrier to resist bacterial infection. β-Defensins are a group of antimicrobial peptides mainly produced by epithelial cells of many organs including skin, lung, kidney, pancreas, uterus, eye, and nasal and oral mucosa. This review focuses on β-defensins (BDs) in oral epithelia and discusses their importance in oral epithelial health and disease. BDs exhibit antimicrobial activity against oral microbes including periodontitis-related bacteria, Candida, and papilloma virus. Alterative expression of BDs was observed in oral epithelial diseases, including oral inflammatory lesions with and without microbial infection and oral cancer. BDs may be useful in the treatment of oral infectious diseases, ulcerative lesions, and cancer. BDs play an important role in protection against oral microbes and may be used in clinical applications.

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.

Defensins in saliva and the salivary glands

Saliva contains several types of antimicrobial peptides that play a role in innate immunity. Peptides that were recently added to this list are the defensins. The purpose of this review is to summarize what is known about the production and role of defensins in the salivary glands and to discuss their therapeutic potential. The Α-defensins, human neutrophil defensins (HNP)-1, -2, and -3, have been detected in saliva and may be derived from neutrophils. The Β-defensins, human Β-defensins (HBD)-1 and -2, have also been detected in saliva. Although it has been speculated that salivary HBDs are derived from keratinocytes that line the oral mucosa rather than from the salivary glands, the HBD-1 peptide was recently found to be specifically expressed in salivary ductal cells, although not in acini. Defensins may be useful for the treatment of periodontal disease and for the prevention of caries and periodontitis.

Pattern of expression of beta-defensins in oral squamous cell carcinoma

Human beta-defensin (hBD)-1 and hBD-2 are antimicrobial peptides that have been detected in certain types of epithelia, including the skin and oral epithelia. It has been suggested that bacterial infection is an important factor in the process of carcinogenesis. The expression of hBDs in oral squamous cell carcinoma (SCC) may be down-regulated. We studied the pattern of expression of hBD-1 and hBD-2 mRNA in oral (SCC) cell lines and in tumor samples obtained from four patients with oral SCC who underwent surgical resection, by reverse transcription-polymerase chain reaction (RT-PCR). Human gingival epithelial (HGE) cells were used as the control. The effect of various inflammatory cytokines on hBD-1 and hBD-2 expression in the HGE cells and SCC cell lines, was also studied. hBD-1 mRNA was detected in the Ca-9, SCC-9 and HSC-4 cell lines, but not in the SAS and KB cell lines. hBD-2 mRNA was detected in all five cell lines. All four tumor samples expressed both hBD-1 and hBD-2 mRNA, although the mRNA level of each protein varied. These results indicate that SCCs in which hBD expression is downregulated, may be susceptible to bacterial infection.

Expression of MIP-3α/CCL20, a macrophage inflammatory protein in oral squamous cell carcinoma

Abstract We have examined the expression of MIP-3α/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3α/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3α, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler™ using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3α mRNA. The expression of MIP-3α was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-α. By in situ hybridization, the detectable MIP-3α expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3α contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

Upregulation of Human Beta-Defensin 2 Peptide Expression in Oral Lichen Planus, Leukoplakia and Candidiasis. An Immunohistochemical Study

Human beta defensin 2 (hBD-2) is a major antimicrobial peptide that is produced by many types of epithelial cells, and is transcriptionally inducible by various proinflammatory agents, such as cytokines and bacteria. Although in vitro studies of the hBDs in oral epithelial cells have been well documented, only little is known about the in vivo pathological state of oral epithelium. We investigated the localization of hBD-2 peptide in tissue sections of oral lichen planus, leukoplakia, candidal leukoplakia and radicular cysts using immunohistochemistry. HBD-2 was stained in both the hyperkeratinized and the granular layers in cases of lichen planus with hyperkeratosis and leukoplakia. Expression in spinous and suprabasal layers was often strong in lichen planus. There were no significant differences in the number of S-100 positive dendritic cells between the widely stained areas and those with limited staining areas in lichen planus. In cases of candidal leukoplakia, the hyphae of candida were mainly detected on the surface of keratinization, which showed only negative or faint staining for hBD-2. These results suggest that hBD-2 is vigorously induced by lichen planus-related inflammation and that it plays an important role in protection from Candida albicans infection; however, it is not a strong chemotactic attractant for Langerhans cells in pathological conditions of oral epithelium.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Expression of inflammatory cytokines and beta-defensin 1 mRNAs in porcine epithelial rests of Malassez in vitro.

In the present study, we investigated the mRNA expression of inflammatory cytokines, including interleukin (IL)-1α, IL-6, IL-8, and granulocyte macrophage colony-stimulating factor (GM-CSF), and β defensin 1 (BD-1), an antimicrobial peptide, in the epithelial rests of Malassez in vitro. A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed in order to observe the expression of these mRNAs. The effect of lipopolysaccharide (LPS) on the mRNA expression was also studied by quantitative RT-PCR assay, with a LightCycler, using the double-stranded DNA dye SYBR Green I. The mRNAs of the four kinds of inflammatory cytokines and BD-1 were detected in the epithelial cells under normal culture conditions. Immunocytochemical staining showed the expression of CD14, a receptor for LPS, on the epithelial cells. The mRNA expressions of IL-1α, IL-6, IL-8, and GM-CSF were upregulated by stimulation with LPS, in a dose- and time-dependent manner. Epithelial cells incubated with 1000 ng/ml of LPS for 6 h showed the most significant upregulation of the cytokine mRNAs. On the other hand, no obvious alteration of BD-1 expression by LPS stimulation was observed. The results indicated that the epithelial rests of Malassez may actively participate in the inflammatory response to bacterial infection, and that they play an important role in the defense mechanism of the radicular cyst.