| 1. |
- Capulli, Mattia, et al.
(författare)
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The C-Terminal Domain of Chondroadherin: A New Regulator of Osteoclast Motility Counteracting Bone Loss
- 2014
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Ingår i: Journal of Bone and Mineral Research. - : Wiley. - 1523-4681 .- 0884-0431. ; 29:8, s. 1833-1846
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Tidskriftsartikel (refereegranskat)abstract
- Chondroadherin (CHAD) is a leucine-rich protein promoting cell attachment through binding to integrin alpha(2)beta(1) and syndecans. We observed that CHAD mRNA and protein were lower in bone biopsies of 50-year-old to 65-year-old osteoporotic women and in bone samples of ovariectomized mice versus gender/age-matched controls, suggesting a role in bone metabolism. By the means of an internal cyclic peptide (cyclicCHAD), we observed that its integrin binding sequence impaired preosteoclast migration through a nitric oxide synthase 2-dependent mechanism, decreasing osteoclastogenesis and bone resorption in a concentration-dependent fashion, whereas it had no effect on osteoblasts. Consistently, cyclicCHAD reduced transcription of two nitric oxide downstream genes, migfilin and vasp, involved in cell motility. Furthermore, the nitric oxide donor, S-nitroso-N-acetyl-D, L-penicillamine, stimulated preosteoclast migration and prevented the inhibitory effect of cyclicCHAD. Conversely, the nitric oxide synthase 2 (NOS2) inhibitor, N5-(1-iminoethyl)-l-ornithine, decreased both preosteoclast migration and differentiation, confirming a role of the nitric oxide pathway in the mechanism of action triggered by cyclicCHAD. In vivo, administration of cyclicCHAD was well tolerated and increased bone volume in healthy mice, with no adverse effect. In ovariectomized mice cyclicCHAD improved bone mass by both a preventive and a curative treatment protocol, with an effect in line with that of the bisphosphonate alendronate, that was mimicked by the NOS2 inhibitor [L-N6-(1-Iminoethyl)-lysine. 2 dihydrochloride]. In both mouse models, cyclicCHAD reduced osteoclast and bone resorption without affecting osteoblast parameters and bone formation. In conclusion, CHAD is a novel regulator of bone metabolism that, through its integrin binding domain, inhibits preosteoclast motility and bone resorption, with a potential translational impact for the treatment of osteoporosis. (C) 2014 American Society for Bone and Mineral Research.
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| 2. |
- Haglund, Lisbet, et al.
(författare)
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Identification and Characterization of the Integrin alpha(2)beta(1) Binding Motif in Chondroadherin Mediating Cell Attachment
- 2011
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286:5, s. 3925-3934
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Tidskriftsartikel (refereegranskat)abstract
- Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin alpha(2)beta(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same alpha(2)beta(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the alpha(2)beta(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.
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| 3. |
- Haglund, Lisbet, et al.
(författare)
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The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains
- 2013
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 288:2, s. 995-1008
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Tidskriftsartikel (refereegranskat)abstract
- Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH(359), now shown to bind to heparin with a K-D of 13 mu M. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their beta(1) integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.
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| 4. |
- Pramhed, Anna, et al.
(författare)
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Purification, crystallization and preliminary X-ray diffraction analysis of human chondroadherin.
- 2008
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 64:Pt 6, s. 516-519
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Tidskriftsartikel (refereegranskat)abstract
- Chondroadherin is a cartilage matrix protein that is known to mediate the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine-rich repeats flanked by cysteine-rich domains at the N- and C-terminal ends. Recombinant human chondroadherin was crystallized using the sitting-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 56.4, b = 111.3, c = 128.5 A, beta = 92.2, and are most likely to contain four molecules in the asymmetric unit. The crystals diffracted to at least 2.3 A using synchrotron radiation, but structure determination using molecular replacement has so far been unsuccessful.
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| 5. |
- Rucci, Nadia, et al.
(författare)
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Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss
- 2013
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Ingår i: Journal of Bone and Mineral Research. - : Wiley. - 1523-4681 .- 0884-0431. ; 28:9, s. 1912-1924
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Tidskriftsartikel (refereegranskat)abstract
- (PRELP)-P-hbd is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (PRELP)-P-hbd inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-B transcription factor activity. In this work, we hypothesized that (PRELP)-P-hbd could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (PRELP)-P-hbd counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (PRELP)-P-hbd reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (PRELP)-P-hbd also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (PRELP)-P-hbd was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (PRELP)-P-hbd could be employed as a novel biological agent to combat experimentally induced bone loss and breast cancer metastases, with a potential translational impact. (C) 2013 American Society for Bone and Mineral Research.
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| 6. |
- Rucci, Nadia, et al.
(författare)
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The α2β1 binding domain of chondroadherin inhibits breast cancer-induced bone metastases and impairs primary tumour growth: A preclinical study.
- 2015
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Ingår i: Cancer Letters. - : Elsevier BV. - 1872-7980 .- 0304-3835. ; 358:1, s. 67-75
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Tidskriftsartikel (refereegranskat)abstract
- cyclicCHAD is a peptide representing the α2β1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact.
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| 7. |
- Tillgren, Viveka, et al.
(författare)
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The tyrosine sulfate rich domains of the LRR-proteins fibromodulin and osteoadherin bind motifs of basic clusters in a variety of heparin binding proteins including bioactive factors.
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; Aug 21, s. 28543-28553
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Tidskriftsartikel (refereegranskat)abstract
- The small leucine-rich repeat proteins (SLRPs), fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N- and C-termini, respectively and PRELP is known to bind to heparin via this domain. Another heparin binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C-terminal. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full length osteoadherin. The tyrosine sulfate domains also bound heparin binding proteins such as basic fibroblast growth factor (FGF-2), thrombospondin I (TSP-I), MMP 13, the NC4 domain of collagen IX and IL10. Fibronectin with large heparin binding domains did not bind, neither did CILP containing a heparin binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10 kDa fragments subfractionated by ion exchange chromatography. These interactions may sequester growth factors, cytokines and MMPs in the extracellular matrix as well as contribute to its organization.
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