Immunohistochemical Study of Amelogenin and Lysosome-Associate Membrane Proteins (LAMPs) in Cartilage

Hatakeyama, Yuji; Hatakeyama, Jun; Oka, Kyoko; Tsuruga, Eichi; Inai, Tetsuichiro; Anan, Hisashi; Sawa, Yoshihiko

doi:10.4067/s0717-95022014000200040

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…Our results showed that several differentiation marker genes in chondrogenic and osteoblast cells were increased by csLRAP and were suppressed in the presence of LAMP-1 antibody. Our previous study suggested that LAMP-1 is involved in chondrogenic differentiation of mesenchymal cells 28) . Thus, our present results suggest that LAMP-1 expression in chondrogenic cells and osteoblast cells is involved in the differentiation process promoted by csLRAP.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, mRNA was reverse-transcribed to cDNA using the iScript TM RT Supermix for RT-qPCR (Bio-Rad, Tokyo, Japan). Real-time PCR was performed using iTaq TM SYBR Green Supermix With ROX (BioRad) for 45 cycles of 95°C for 30 seconds, 60°C for 45 seconds, and 72°C for 30 seconds, using specific primers designed for osteogenic and chondrogenic differentiation marker genes as previously reported 13,[28][29][30] . Mouse Col1a2 specific primers were designed using National Center for Biotechnology Information .…”

Section: Immunofluorescence Assaymentioning

confidence: 99%

“…After washing with PBS, cells were incubated with rabbit polyclonal anti-mouse LAMP-1 (ab24170, Abcam, Tokyo Japan) diluted to 1 g/ml in 1% bovine serum albumin (BSA) for one hour at room temperature. After incubation with the primary antibody, cells were incubated with goat anti- 28) . Briefly, mRNA was reverse-transcribed to cDNA using the iScript TM RT Supermix for RT-qPCR (Bio-Rad, Tokyo, Japan).…”

Section: Immunofluorescence Assaymentioning

confidence: 99%

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Effects of a Chemically Synthesized Leucine-Rich Amelogenin Peptide (csLRAP) on Chondrogenic and Osteogenic Cells

Matsuda

Hatakeyama

Nakashima

et al. 2017

J. Hard Tissue Biology.

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Amelogenin is one of the enamel matrices secreted by ameloblasts and is known to have at least 15 alternative mRNA splicing isoforms. The leucine rich amelogenin peptide (LRAP) is one of most abundant amelogenin isoforms and numerous studies have shown that amelogenin peptides including LRAP, are expressed in cartilage and bone. Lysosome-associated membrane protein-1 (LAMP-1) has been identified as amelogenin binding partner, however, the mechanism of action for LRAP in chondrogenesis or osteogenesis is still unclear. This study aimed to assay the effect of chemically synthesized LRAP (csLRAP) on chondrogenic or osteogenic differentiation with the involvement of LAMP-1. The csLRAP used in this study was generated by F-moc solid phase chemistry based on the mouse LRAP amino acid sequence and was added to the culture medium of the chondrogenic cells, ATDC5 and the osteoblast cells, MC3T3-E1. Both chondrogenic and osteoblast cells, in which an immunopositive reaction to LAMP-1 antibody was observed by immunocytochemistry, showed significant suppression in cell number in the presence of csLRAP at 10 g/ml compared to that in control, but this effect was rescued in the presence of LAMP-1 antibody. On the other hand, the intensity of alcian blue staining in chondrogenic cells and alizarin red staining in osteoblast cells was significantly increased in the presence of csLRAP at a dose of 10 g/ml after 4 weeks culture, and this effect was suppressed in the presence LAMP-1 antibody. Moreover, the chondrogenic or osteogenic differentiation marker genes including Sox9, Type II Collagen, Type X Collagen, Runx2, Alkaline Phosphatase, and Type I Collagen, were upregulated in the presence of csLRAP after one week of culture and were suppressed in the presence of LAMP-1 antibody. These results suggest that csLRAP could promote osteogenic and chondrogenic differentiation in vitro, and that LAMP-1 may be involved in the differentiation and proliferation of these cells.

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Section: Discussionmentioning

confidence: 99%

Section: Immunofluorescence Assaymentioning

confidence: 99%

Section: Immunofluorescence Assaymentioning

confidence: 99%

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Effects of a Chemically Synthesized Leucine-Rich Amelogenin Peptide (csLRAP) on Chondrogenic and Osteogenic Cells

Matsuda

Hatakeyama

Nakashima

et al. 2017

J. Hard Tissue Biology.

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“…Tissue preparation and immunofluorescence staining were performed according to previously reported methods with minor modifications (Hatakeyama et al, 2014). WT and Amelx* mice (10-week-old) were anesthetized and perfused with 4 % PFA in 0.1M PBS, pH 7.4.…”

Section: Tissue Preparation and Immunofluorescence Stainingmentioning

confidence: 99%

Immunohistochemical Study of Amelogenin Binding Proteins in an Amelogenin Point Mutation Mouse

et al. 2019

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OTAWA-KAMOGASHIRA, N.; MATSUDA, Y.; TAKEZAKI, M.; HATAKEYAMA, Y.; TAMAOKI, S. & ISHIKAWA, H.Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse. Int. J. Morphol., 37(2):522-532, 2019. SUMMARY:Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts. KEY WORDS: Amelogenin; Amelogenesis imperfecta; 78kDa glucose-related protein (Grp78); Lysosome-associated membrane proteins (LAMPs). OTAWA-KAMOGASHIRA, N.; MATSUDA, Y.; TAKEZAKI, M.; HATAKEYAMA, Y.; TAMAOKI, S. & ISHIKAWA, H. Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse. OTAWA-KAMOGASHIRA, N.; MATSUDA, Y.; TAKEZAKI, M.; HATAKEYAMA, Y.; TAMAOKI, S. & ISHIKAWA, H. Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse. Int. J. Morphol., 37(2):527-537, 2019.

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“…), and in the epiphyseal growth plate (Hatakeyama et al. ). Reports suggesting that lysosome‐associated membrane protein 1 (Lamp‐1), a transmembrane protein, serves as a cell surface binding receptor for full length amelogenin and leucine‐rich amelogenin peptide (LRAP), a small amelogenin gene‐spliced product, indicate a possibility that amelogenin may function as a paracrine or autocrine molecule in cell signaling (Tompkins et al.…”

Section: Introductionmentioning

confidence: 99%

Expression of amelogenin and effects of cyclosporin A in developing hair follicles in rats

Yoo

Lee

et al. 2015

Journal of Anatomy

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Amelogenin, an enamel matrix protein has been considered to be exclusively expressed by ameloblasts during odontogenesis. However, burgeoning evidence indicates that amelogenin is also expressed in non-mineralizing tissues. Under the hypothesis that amelogenin may be a functional molecule in developing hair follicles which share developmental features with odontogenesis, this study for the first time elucidated the presence and functional changes of amelogenin and its receptors during rat hair follicle development. Amelogenin was specifically localized in the outer epithelial root sheath of hair follicles. Its expression appeared in the deeper portion of hair follicles, i.e. the bulbar and suprabulbar regions rather than the superficial region. Lamp-1, an amelogenin receptor, was localized in either follicular cells or outer epithelial sheath cells, reflecting functional changes during development. The expression of amelogenin splicing variants increased in a time-dependent manner during postnatal development of hair follicles. Amelogenin expression was increased by treatment with cyclosporin A, which is an inducer of anagen in the hair follicle, whereas the level of Lamp-1 and -2 was decreased by cyclosporin A treatment. These results suggest that amelogenin may be a functional molecule involved in the development of the hair follicle rather than an inert hair shaft matrix protein.

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Immunohistochemical Study of Amelogenin and Lysosome-Associate Membrane Proteins (LAMPs) in Cartilage

Cited by 5 publications

References 28 publications

Effects of a Chemically Synthesized Leucine-Rich Amelogenin Peptide (csLRAP) on Chondrogenic and Osteogenic Cells

Effects of a Chemically Synthesized Leucine-Rich Amelogenin Peptide (csLRAP) on Chondrogenic and Osteogenic Cells

Immunohistochemical Study of Amelogenin Binding Proteins in an Amelogenin Point Mutation Mouse

Expression of amelogenin and effects of cyclosporin A in developing hair follicles in rats

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