Transcription factor EB influences invasion and migration in oral squamous cell carcinomas.

OBJECTIVE: Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and plays an important role in various cancers. However, the function of TFEB in oral squamous cell carcinomas has not been examined. The aim of this study was to elucidate the role of TFEB in oral squamous cell carcinomas. MATERIALS AND METHODS: Expression levels of TFEB were examined in six different human oral squamous carcinoma cells: HSC2, HSC3, HSC4, SAS, OSC20, and SCC25. Knockdown of TFEB using small interfering RNA in HSC2 and HSC4 cells was performed. Cell morphology was observed by immunofluorescence microscopy. Cell proliferation, invasion, and adhesion were analyzed. RESULTS: Expression levels of TFEB were high in HSC2, moderate in HSC4 and SCC25, and low in HSC3 and OSC20 cells. Knockdown of TFEB did not affect proliferation of HSC2 and HSC4 cells, but did induced enlargement of lysosomes and endosomes in HSC4 cells. TFEB silencing reduced invasion and migration of these HSC cell squamous carcinoma cells; however, increased cell adhesion was also observed. CONCLUSION: TFEB knockdown reduces invasion and migration of cancer cells, likely through lysosomal regulation. Taken together, TFEB influences cell invasion and migration of oral squamous cell carcinomas.

Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas in vitro.

OBJECTIVE: Cathepsin K was initially discovered as an osteoclast-specific cysteine proteinase, but the enzyme is also expressed in various cancers including oral squamous cell carcinomas. This study aimed to clarify the function of cathepsin K in oral squamous cell carcinomas. MATERIALS AND METHODS: Expression levels of cathepsin K were examined in six types of cell carcinomas. Carcinomas overexpressing cathepsin K were constructed. Effects of cathepsin K overexpression and treatment with odanacatib, a specific cathepsin K inhibitor, on cell invasion, migration and adhesion were analysed. RESULTS: Different levels of cathepsin K were expressed in carcinomas. Cathepsin K was predominantly localised in lysosomes. Cathepsin K overexpression impaired the proliferation of carcinomas. Invasion analysis showed that cathepsin K overexpression enhanced invasion and migration of carcinomas, whereas inhibition of cathepsin K by odanacatib caused the opposite effects in carcinomas. Cathepsin K overexpression also increased cell adhesion and slightly increased surface expression of the adhesion receptor CD29/integrin ß1 . CONCLUSIONS: The enhanced invasion of carcinomas resulting from cathepsin K overexpression is probably due to the increased cell migration and adhesion. Thus, cathepsin K is implicated not only in protein degradation but also in invasion, migration and adhesion of oral squamous cell carcinomas.

Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells.

AIM: To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells. METHODOLOGY: Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test. RESULTS: The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively. CONCLUSIONS: These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

Immunohistochemical localization of cathepsins D and E in human gastric cancer: a possible correlation with local invasive and metastatic activities of carcinoma cells.

The immunohistochemical localization of cathepsins D and E in 44 cases of human gastric carcinoma, using antibodies specific for each enzyme, were investigated. Cathepsin D- and E- positive carcinoma cells were present in all samples. However, the staining intensity varied from cell to cell in the same carcinoma tissue as well as among samples. The most intense immunostaining of both cathepsins was often found in the cells, which were present at the advancing margin of the carcinoma tissues. The incidence of this peculiar localization of intensely stained carcinoma cells significantly correlated with the progression of the carcinoma tissue (D, P < .05; E, P < .01) and with occurrence of the lymph node metastasis (D and E, P < .05). There was no statistical significance between this localization and the histological type (differentiation) of the carcinoma tissues. Cathepsin-positive inflammatory cells infiltrated in and around the carcinoma tissue, and intensely stained inflammatory cells were often located in the stroma at the border of the carcinoma tissue. However, no statistical correlation was noted between the localization of cathepsin-positive inflammatory cells at the border and the stage of progression or the incidence of metastasis. These results indicated that cathepsins D and E in the carcinoma cells located at the advancing margin play an important role in the invasion and subsequent metastasis of human gastric carcinoma. Meanwhile, cathepsin-positive inflammatory cells seem to be less responsible for the biological behavior of carcinoma cells than those in the carcinoma cells themselves.

Biochemical properties of the monomeric mutant of human cathepsin E expressed in Chinese hamster ovary cells: comparison with dimeric forms of the natural and recombinant cathepsin E.

Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43 ->Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37 degrees C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.

New functional aspects of cathepsin D and cathepsin E.

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.

Immunocytochemical localization of cathepsin D in rat junctional epithelium.

Localization of cathepsin D was studied in the junctional epithelium (JE) of healthy rat gingivae by immuno-light and -electron microscopy, by means of both the avidin-biotin-peroxidase complex method and a colloidal gold IgG method. At the light-microscopic level, cathepsin D was demonstrated in the JE and oral sulcular epithelium (OSE). Cathepsin D immunoreactivity was remarkable in the coronal portion of the JE and decreased toward its apical portion. However, cathepsin D immunoreactivity in the basal cell layer of the JE was negligible or negative. In the OSE, the granular layer was positive for cathepsin D. In the adjacent connective tissue, many macrophage-like cells (not clear at this level) close to the basal cell layer showed strong immunoreactivity. At the electron microscopic level, cathepsin D was found in the primary lysosomes and trans-cisternae of Golgi apparatus in the JE cells. These lysosomes were often fused together or were fused with cathepsin D-negative intracytoplasmic vacuoles to form secondary lysosomes, which indicated that intracellular digestion may have been in progress. However, neutrophils contained few gold particles based on cathepsin D. It is likely that the amounts of cathepsin D contained in the JE cells and macrophages are larger than those of cathepsin D contained in the neutrophils. These findings provided morphological evidence that JE cells have the same endocytotic capacity as macrophages and neutrophils, and that JE cells participate in the intracellular digestion that is carried out by lysosomal enzymes such as cathepsin D. It is suggested, in addition, that maximum intracellular digestion occurs in the coronal portion of the JE.

Immunohistochemical localization of cathepsins B and D in the synovial lining cells of the normal rat temporomandibular joint.

In 1 micron-thick serial cryosections, cathepsins B and D were found to coexist in both type A (macrophage-like) and type B (fibroblast-like) cells: the whole cytoplasm of the type A cells showed strong immunoreactivity, while the type B cells contained a few granular reaction products. It is therefore suggested that type A cells have a marked ability for intracellular digestion of organic materials.

Immunocytochemical localization of cathepsin L in the synovial lining cells of the rat temporomandibular joint.

Localization of cathepsin L in the synovial lining cells of the normal rat temporomandibular joint was investigated by the avidin-biotin-peroxidase complex method for semithin (1 microns) cryosections and the colloidal gold-labelled IgG method for ultrathin sections of LR gold resin. At the light-microscopic level, type A (macrophage-like) and B (fibroblast-like) cells formed the synovial lining layer. Extensive immunoreactivity for cathepsin L was observed in many granules and vacuoles of type A cells, while in the type B cells, immunoreactivity was found in very few granules. In the sublining layer, macrophages and a few fibroblasts were positive for cathepsin L. By electron microscopy, at the peripheral cytoplasm of the type A cells close to the lateral intercellular spaces and joint cavity, numerous coated vesicles and vacuoles (probably early endosomes) indicating endocytotic function were found. Gold particles indicating cathepsin L were localized in the vesicles (primary lysosomes) in the perinuclear cytoplasm and in the larger amorphous vacuoles (1 microns dia) as phagolysosomes. In type B cells, gold particles were limited to the vesicles only (primary lysosomes). The cathepsin L-positive primary lysosomes were numerous in a few fibroblasts in the sublining layer. These results indicate that type A cells contain a large amount of cathepsin L, and suggest that these cells endocytose surplus substances such as collagen and proteoglycan fragments in normal rat TMJ, effecting their digestion and degradation by the action of this proteolytic cathepsin.

Role of the COOH-terminal domains of meprin A in folding, secretion, and activity of the metalloendopeptidase.

Secreted forms of the alpha subunit of recombinant mouse meprin A include an NH2-terminal prosequence, a catalytic domain, and three COOH-terminal domains designated as MAM (meprin, A-5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (after MATH). In this study, the importance of these COOH-terminal domains for biosynthesis of secreted, activable forms of the protease was investigated. Transcripts of the meprin subunit truncated after the protease (alpha(1-275)), MAM (alpha(1-452)), and MATH (alpha(1-528)) domains or with individual domains deleted (DeltaMAM, DeltaMATH, and DeltaAM), were transfected into human embryonic kidney 293 cells. The wild-type subunit, DeltaMATH, DeltaAM, alpha(1-452), and alpha(1-528) were secreted into the media, although the DeltaAM mutant was secreted at very low levels. The DeltaMATH and alpha(1-452) mutants were not activable by limited proteolysis. The alpha(1-528) mutant was as active as wild-type meprin alpha against a bradykinin substrate, but had no activity against azocasein, and it, as all other mutants, was more vulnerable to extensive degradation by proteases than the wild-type protein. Pulse-chase experiments revealed that the DeltaMAM and alpha(1-275) mutants were rapidly degraded within cells. Treatment with lactacystin, a specific inhibitor of the proteasome, significantly decreased the degradation, indicating that the mutants lacking the MAM domain are degraded by the proteasome as misfolded proteins. These results indicate that the MAM domain is necessary for correct folding and transport through the secretory pathway, the MATH domain is required for folding of an activable zymogen, and the AM domain is important for activity against proteins and efficient secretion of the protein. The work demonstrates the interdependence of the domains for correct folding of an activable, stable, mature enzyme.

Expression and localization of a major lysosomal membrane sialoglycoprotein (LGP107) in plasma membranes of rat osteoblasts and osteocytes.

The immunocytochemical localization of a major lysosomal membrane sialoglycoprotein with a molecular mass of 107 kDa, which was designated as LGP107, was investigated in osteoblast lineage cells involved in osteoclastic bone resorption using specific polyclonal antibody against LGP107. Osteoclastic bone resorption was induced by transplantation of parathyroid glands. In control experiments, no immunoreaction product for LGP107 was recognized in osteoblasts and osteocytes. Strong immunoreaction products for LGP107 occurred on the plasma membranes in the osteoblasts and osteocytes prior to the appearance of osteoclasts one day after transplantation of the parathyroid glands. Furthermore, two days after induction, strong diaminobenzidine reactions were also observed on the plasma membranes in the osteoblastic cells adjacent to the active osteoclasts. These data suggest that LGP107 in osteoblastic cells and osteocytes may play an important role in cell-recognition and/or cell-adhesion, and that LGP107 may be involved in osteoblastic degradation of the osteoid as well as exposure of the bone surface.

Immunohistochemical localization of cathepsins B, D and L in the rat osteoclast.

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-micron-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathepsin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.

Transient forebrain ischemia induces increased expression and specific localization of cathepsins E and D in rat hippocampus and neostriatum.

The accumulation and localization of cathepsins E and D in the rat hippocampus and neostriatum during the neurodegenerating process induced by transient forebrain ischemia were investigated by immunoprecipitation and by immunohistochemistry using discriminative antibodies specific for each enzyme. While significant amounts of cathepsin D were found in both the hippocampus and the neostriatum of normal rats, cathepsin E was barely detectable in these tissues. No significant change in their levels was found in these tissues of postischemic rats for up to 3 days after transient forebrain ischemia. After 7 days of the treatment, cathepsin E was markedly increased in both tissues. Although the cathepsin D content in these tissues was also increased at this stage, the rate of increase was much less than that of cathepsin E. At the light microscopic level, the increased immunoreactivity for each enzyme was mainly found in reactive glial cells and degenerating neurons in the hippocampal CA1 subfield at 7 days postischemia. In the neostriatal dorsolateral portion, cathepsin D immunoreactivity was also increased in both reactive glial cells and degenerating neurons, whereas increased immunoreactivity of cathepsin E was only identified in reactive glial cells at 7 days postischemia. It was also found by double-immunostaining technique that the cathepsin E-positive glial cells were largely reactive microglial cells, whereas the cathepsin D-positive glial cells were associated mainly with reactive astrocytes. These results suggest that the accumulation of both cathepsins E and D in the regions of selective neuronal vulnerability may be associated with the postischemic development of intense gliosis and also probably neurodegenerative responses.

Increased synthesis and specific localization of a major lysosomal membrane sialoglycoprotein (LGP107) at the ruffled border membrane of active osteoclasts.

The immunocytochemical localization was investigated of a major lysosomal membrane sialoglycoprotein with a molecular mass of 107 kDa, which was designated as LGP107. The study utilized rat osteoclasts with different bone resorbing activity and osteoclast precursors at various stages of differentiation and maturation together with monospecific antibodies to this protein. Despite its localization primarily in lysosomes and endosomes in the other cell types examined, LGP107 was exclusively confined to the apical plasma membrane at the ruffled border of the active osteoclast, where the osteoclast is in contact with the bone surface. The protein was also concentrated in a number of endocytic vacuoles in the vicinity of the ruffled border membrane. However the labeling was not found in the basolateral membranes of the active osteoclast. The ruffled border membrane detached from the bone surface showed a marked decrease in the extent of the immunolabeling. The post- and/or resting osteoclasts, which were located away from the bone surface, were totally devoid of the membraneous localization of LGP107. No definite immunolabeling was found in the immature preosteoclasts. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane by vectorial vesicle transport. LGP107 is suggested to contribute to the formation and maintenance of the specialized acidic environment for bone resorption.

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts.

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.

N-Linked oligosaccharides on the meprin A metalloprotease are important for secretion and enzymatic activity, but not for apical targeting.

The alpha and beta subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; approximately 25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin alpha and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin alpha. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75-100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin alpha (alpha-(1-445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin alpha.

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis.

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene (rgp1) and was also present in genes for lysine-specific cysteine proteinases (prtP and kgp) and a haemagglutinin (hagA) of P. gingivalis. The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis.