Biomedical subjects

M Enomoto-Iwamoto

Publications and source records attributed to M Enomoto-Iwamoto.

8 recordsLinked to original sources

PubMed1999

Vinculin, Talin, Integrin alpha6beta1 and laminin can serve as components of attachment complex mediating contraction force transmission from cardiomyocytes to extracellular matrix.

Recently, we reported that cardiomyocytes adhere to extracellular matrix at costameres, the striated distribution of vinculin between Z-lines and the sarcolemma, where transmission of contraction forces from myofibrils to the extracellular matrix occurs. To identify other molecules involved in force transmission at costameres, we examined adult rat and embryonic chick cardiomyocytes cultured on coverslips or flexible thin silicone rubber substrata. Immunolocalization of talin showed a costameric, striated distribution, which corresponded to dark contacts with interference reflection microscopy. The molecules involved in substrate adhesion were cross-linked with the non-penetrating cross-linking agent Bis(sulfosuccinimidyl)-suberate and detected by immunohistochemical staining with anti-alpha6, alpha3, alphav, or beta1 integrin antibodies. Both alpha6 and beta1 showed costameric distributions, but alpha3 and alpha(v) did not. The distribution of laminin after cross-linking and extraction also showed a costameric distribution. When anti-integrin beta1 antibody was added to live cardiomyocytes grown on the silicone rubber substratum, the transmission of contraction forces was inhibited. These findings suggest that vinculin, talin, integrin alpha6beta1 and laminin system can be involved in transmission of contraction force to the extracellular matrix.

Animals↗

PubMed1998

Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes.

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.

Animals↗

PubMed1997

Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation.

We investigated the role of Sonic hedgehog (SHH) in osteoblast differentiation and bone formation. The numbers of ALP-positive cells in the mouse fibroblastic cell line C3H10T1/2 and the mouse osteoblastic cell line MC3T3-E1 were increased by co-culture with chicken fibroblasts transfected with chicken Shh cDNA encoding amino-terminal peptide (Shh-N). The conditioned medium of Shh-N-RCAS-transfected chicken fibroblast cultures also significantly increased ALP activity in both C3H10T1/2 and MC3T3-E1 cells. Intramuscular transplantation of Shh-N-RCAS-transfected chicken fibroblasts into athymic mice induced ectopic bone formation. These results indicate that SHH induces osteoblast differentiation and ectopic bone formation.

3T3 Cells↗

PubMed1997

Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes.

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of alpha 5 beta 1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on alpha 5 beta 1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against alpha 5 beta 1 or fibronectin, indicating that cell spreading involved alpha 5 beta 1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with alpha 5 beta 1 integrin-ligand interactions. Furthermore, DNA content was decreased by treatment with anti-fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the alpha 5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that alpha 5 beta 1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte-matrix interactions and proliferation.

Animals↗

PubMed1996

BMP signaling during bone pattern determination in the developing limb.

To examine the role of BMP signaling during limb pattern formation, we isolated chicken cDNAs encoding type I (BRK-1 and BRK-2) and type II (BRK-3) receptors for bone morphogenetic proteins. BRK-2 and BRK-3, which constitute dual-affinity signaling receptor complexes for BMPs, are co-expressed in condensing precartilaginous cells, while BRK-1 is weakly expressed in the limb mesenchyme. BRK-3 is also expressed in the apical ectodermal ridge and interdigital limb mesenchyme. BRK-2 is intensely expressed in the posterior-distal region of the limb bud. During digit duplication by implanting Sonic hedgehog-producing cells, BRK-2 expression is induced anteriorly in the new digit forming region as observed for BMP-2 and BMP-7 expression in the limb bud. Dominant-negative effects on BMP signaling were obtained by over-expressing kinase domain-deficient forms of the receptors. Chondrogenesis of limb mesenchymal cells is markedly inhibited by dominant-negative BRK-2 and BRK-3, but not by BRK-1. Although the bone pattern was not disturbed by expressing individual dominant-negative BRK independently, preferential distal and posterior limb truncations resulted from co-expressing the dominant-negative forms of BRK-2 and BRK-3 in the whole limb bud, thus providing evidence that BMPs are essential morphogenetic signals for limb bone patterning.

Amino Acid Sequence↗

PubMed1995

Regulation of integrin alpha 5 beta 1 affinity during myogenic differentiation.

The antigen recognized by U1 alpha, a monoclonal antibody to the alpha chain of a chicken integrin fibronectin receptor, was identified as alpha 5. It identifies the same polypeptide as antisera raised to a sequence from the alpha 5 cytoplasmic domain. The U1 alpha antibody has the unusual functional property for alpha chain antibodies of enhancing the binding of alpha 5 beta 1 for its ligand fibronectin. U1 alpha was used to examine the function of alpha 5 beta 1 during myogenic differentiation. As myogenic cells differentiated from replicating myoblasts to bipolar myocytes there was a decrease in their adhesion to the substrate caused by inactivation of alpha 5 beta 1, which could be reversed by treatment of the cells with U1 alpha. The U1 alpha induced increased adhesion to fibronectin but did not inhibit the differentiation process as measured by formation of myotubes. However, U1 alpha did interfere with both cell migration and morphogenesis of myotubes. The resulting myotubes were smaller, more branched, and showed less regular alignment of nuclei. The results suggest that the ability of the cell to regulate alpha 5 beta 1 affinity is critical to myogenic morphogenesis.

Animals↗

PubMed1995

Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.

Animals↗

PubMed1993

Evaluation of integrin molecules involved in substrate adhesion.

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.

3T3 Cells↗