Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system.

Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis.

Cathepsin expression in oral squamous cell carcinoma: relationship with clinicopathologic factors.

OBJECTIVE: Proteases are involved in the invasion and metastasis of carcinoma cells. In vivo, oral carcinoma cells easily invade the bone tissue and metastasize to the submandibular and neck lymph nodes. Cathepsin expression has been shown in some neoplastic tissues and serves as a prognostic indicator. The purpose of this study was to investigate the relationship between clinicopathohistologic grades and cathepsin expressions in oral squamous cell carcinoma and to investigate which cathepsin provides prognostic information for patients with oral carcinoma. STUDY DESIGN: Immunohistochemical studies were performed on 78 carcinoma samples with monoclonal antibodies against cathepsins B, H, and L, and a polyclonal antibody against cathepsin D. Serial sections were stained by hematoxylin-eosin staining and classified by Anneroth's classification. Cathepsin B, H, L and D activities of blood serum were determined. Positive results indicative of the presence of cathepsin were investigated to determine any correlation between a particular cathepsin and histologic malignancy grades, tumor cell growth, serum cathepsin activities, and clinical factors. RESULTS: Cathepsins B, H, L, and D were positive in every case. Although the labeling indices for cathepsins B (CB-LI), H (CH-LI), and D (CD-LI) for the cancer cases showed significant differences from those of controls, cathepsin L (CL-LI) of cancer cases showed no difference from that of controls (P <.05). A close correlation was found between CD-LI and T categories of TNM classification (P <.05), and between CD-LI and PCNA-LI (P <.05). Furthermore, a close correlation was found between CD-LI and N categories in TNM classification (P <.05). Pathologically, a close correlation was found between CB-LI or CD-LI and the pattern and/or stage of invasion (P <.05). CONCLUSION: Cathepsin D and B expression were closely correlated with carcinoma invasion and progression. These proteases may be useful in determining the prognoses of patients with oral carcinoma.

Expressão de TGFβ1 mRNA nas fases iniciais de expansão da sutura palatina mediana / The expression of TGFβ1 mRNA in the early stage of the midpalatal suture cartilage expansion

INTRODUÇÃO: a expansão da maxila induz a formação de novo osso na sutura palatina mediana por um processo de proliferação e diferenciação celular. A força de expansão pode estimular, nas células progenitoras, a produção de citocinas com atividade osteoindutiva, tais como o transforming growth factor β1(TGFβ1). OBJETIVOS: o principal objetivo deste estudo foi determinar a função dessa citocina nos estágios iniciais de expansão da sutura palatina mediana. MÉTODOS: um aparelho ortodôntico foi instalado entre os molares superiores direito e esquerdo de ratos com 4 semanas de idade. A força de expansão inicial foi de 50g. Os grupos controle e experimental foram sacrificados nos dias 0, 2 e 5. Cortes bucais de 6µm foram obtidos e sujeitos à técnica de hibridização in-situ. RESULTADOS: dois dias após a aplicação de força, as células osteocondroprogenitoras, distribuídas no lado interno do tecido cartilaginoso, exibiram altos níveis de transcrição de transforming growth factor β1. No dia 5, o nível de transcrição de TGFβ1 foi observado nos osteócitos e nas células osteoblásticas, na superfície do novo osso. A atividade osteoblástica foi confirmada por meio de um estudo imunohistoquímico utilizando-se Osteocalcina-Pro (OC-Pro). CONCLUSÕES: os dados sugerem que a expansão da sutura palatina induz a diferenciação de células osteocondroprogenitoras em osteoblastos, estimuladas pela produção de citocinas.

INTRODUCTION: The application of an orthodontic expansion force induces bone formation at the midpalatal suture because of cell proliferation and differentiation. Expansion forces may stimulate the production of osteoinductive cytokines, such as transforming growth factor β1 (TGFβ1), in the progenitor cells. OBJECTIVES: This study determined the role of TGFβ1 in the early stage of midpalatal suture cartilage expansion. METHODS: A rectangular orthodontic appliance was placed between the right and left upper molars of 4-week-old rats. The initial expansion force was 50 g. Animals in the control and experimental groups were sacrified on days 0, 2, and 5 and 6 µmm thick sections were prepared for an in situ hybridization technique. RESULTS: Two days after the application of force, prechondroblastic and undifferentiated mesenchymal cells distributed along the inner side of the cartilaginous tissue had high levels of TGFβ1 transcription. On day 5, the TGFβ1 transcription was found in osteocytes and osteoblastic cells on the surface of newly formed bone. Immunohistochemistry using Osteocalcin-Pro (OC-Pro) confirmed osteoblastic activity. Conclusions: Results suggest that the expansion of midpalatal suture cartilage induces differentiation of osteochondroprogenitor cells into osteoblasts after stimulation by cytokine production.

An involvement of granulocyte medullasin in phenytoin-induced gingival overgrowth in rats.

To investigate the relationship between histological changes and distributions of medullasin, a neutrophil elastase-like serine proteinase, in phenytoin-induced gingival overgrowth, we established a rat model of gingival overgrowth. Thirty-two, 20-day-old male Fischer 344 rats were fed a diet containing phenytoin and sacrificed at 1, 2, 4 and 8 weeks. Control rats (n = 40) were fed the same diet, but without the drug and killed at the same weeks as experimental rats (n = 32) and 0 week (n = 8). The mandible specimens were resected and sectioned bucco-lingually between the first and second molars. A marked inflammatory-cell infiltration and elongated rete pegs were seen in the phenytoin-treated group. The extent of the overgrowth assessed by computer image analysis and the density of medullasin-positive cells by immunohistochemistry in the approximal gingiva showed a significant increase in the phenytoin-treated group compared to the control group. A marked infiltration of the positive cells in experimental rats was observed as early as 2 weeks when gingival overgrowth was not fully established. Medullasin-positive cells were mostly neutrophils and partly macrophage-like cells. These findings suggest that medullasin may be involved in mainly host defense and secondarily collagen metabolism in the phenytoin-induced rat model of gingival overgrowth.

The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin k.

Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.