Taishin Takuma

Fluid Shear Stress-induced Cyclooxygenase-2 Expression Is Mediated by C/EBP β, cAMP-response Element-binding Protein, and AP-1 in Osteoblastic MC3T3-E1 Cells

Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5′-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, −172 to −100 base pair (bp) and −79 to −46 bp, of the cox-2 promoter, in which putativecis-elements of C/EBP β, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP β, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP β and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP β, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts.

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.

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.

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.

Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions ; immunohistochemistry and in situ hybridization

Abstract. Human β-defensin(hBD)-2 , an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.

Interaction of SNARE proteins in rat parotid acinar cells.

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+‐ and phospholipid‐dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose‐response studies, PMA stimulated amylase release independently of db‐cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic‐cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.

Effects of thyroxine and 5α-dihydrotesterone on the activities of various enzymes in the mouse submandibular gland

Abstract The effects of thyroxine and 5α-dihdrrotesterone on the activities of various enzymes in the submandibular gland of young female mice were compared with the following results. 1. 1) Trypsin-like esteroprotease was induced as much by thyroxine as by 5α-dihydrotesterone, and the effects of the two hormones were additive. 2. 2) Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was induced much more by thyroxine than by 5α-dihydrotestosterone, but when 5α-dihydrotestosterone of both hormones were injected daily for more than six days the activity decreased toward the initial level. 3. 3) Mitochondrial α-glycerophosphate dehydrogenase (EC 1.1.99.5) was induced by thyroxine, but 5α-dihydrotestosterone slightly suppressed the induction by thyroxine. 4. 4) The effects of thyroxine on these three enzymes were dose-dependent. 5. 5) Thyroxine induced the three enzymes in ovariectomized and adrenalectomized mice as well as in intact mice. 6. 6) 5α-Dihydrotestosterone caused slight but significant induction of protease and glucose-6-phosphate dehydrogenase in thyroidectomized mice. These results show that thyroxine affects the submandibular gland directly and independently of 5α-dihydrotesterone and that the two hormones have synergitic actions.

In Vitro effects of thyroxine and insulin on myoblasts from chick embryo skeletal muscle

Abstract The role of insulin and l -thyroxine (L-T4) in stimulating myoblast proliferation and differentiation was investigated in vitro. A superphysiological concentration of insulin or a physiological concentration of L-T4 was added to cultures of myoblasts from 11-day-old chick embryo thigh muscle, grown in serum-free DM-153 medium. While the addition of insulin resulted in an increase in the total number of cells, in the extent of fusion, and in the creatine phosphokinase (CPK) activity, myotubes changed into globular structures which tended to degenerate rapidly. On the other hand, while the addition of L-T4 had less effect on myogenesis, myotubes retained their differentiated state longer. Furthermore, the two hormones exhibited synergistic effects. An increase in the initial cell density resulted in an increase in the amount of protein and CPK activity, irrespective of the presence or absence of the hormones. This suggests that the effect of insulin and L-T4 on myogenesis is not a differentiation-specific effect, but rather an indirect result of cell proliferation.

Upregulated expression of human β defensin-1 and -3 mRNA during differentiation of keratinocyte immortalized cell lines, HaCaT and PHK16-0b

BACKGROUND Human beta-defensins (hBDs) belong to a group of antimicrobial peptide that are expressed in the epithelial cells. OBJECTIVE The present study investigated mRNA expression levels of the beta-defensins, hBD-1, -2 and -3, in human keratinocytes during differentiation in vitro. METHODS Immortalized keratinocyte cell lines, HaCaT and PHK16-0b, were used in this study; in order to stimulate differentiation, the Ca(2+) concentration in the growth media was increased from 0.3 to 1.8 mM. RESULTS Four days after the increase, the expression levels of hBD-1 and -3 were increased in both cell lines, followed by an increase in the mRNA levels of the differentiation markers, involucrin and keratin 10. No increased expression of hBD-2 was observed. CONCLUSION The results indicate that keratinocyte differentiation may stimulate hBD-1 and -3 expression in stratified squamous epithelia.