A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures

Hong, Hsiang‐Hsi; Pischon, Nicole; Santana, Ronaldo B.; Palamakumbura, Amitha H.; Chase, Hermik Babakhanlou; Gantz, Donald; Guo, Ying; Üzel, Mehmet; Ma, Daniel; Trackman, Philip C.

doi:10.1002/jcp.10476

Cited by 72 publications

(100 citation statements)

References 53 publications

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“…[30][31][32] However, it remains unclear if dexamethasone can selectively induce apoptosis of some populations of bone marrow cells which were thought to have poor differentiation capability as suggested by Oshina et al 33 or if glucocorticoids fail to induce terminal osteoblast differentitation. 34 Our results also corresponded to Hong et al 35 who reported an early expression but a late secretion and deposition of collagen I in osteoblast cultures. We found significant peaks of collagen I in DAG and DAG þ BMP-2-stimulated cultures but not in controls at day 28, whereas the collagen I levels in culture media showed no significant differences at days 4 and 14.…”

Section: Discussionsupporting

confidence: 91%

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Jäger

Fischer

Dohrn

et al. 2008

Journal Orthopaedic Research

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Dexamethasone/ascorbic acid/glycerolphosphate (DAG) and bone morphogenic protein (BMP)-2 are potent agents in cell proliferation and differentiation pathways. This study investigates the in vitro interactions between dexamethasone and BMP-2 for an osteoblastic differentiation of mesenchymal stem cells (MSCs). Bone marrow-derived human MSCs were cultured with DAG (group A), BMP-2 þ DAG (group B), and DAG þ BMP-2 combined with a porous collagen I/III scaffold (group C). RT-PCR, ELISA, immuncytochemical stainings and flow cytometry analysis served to evaluate the osteogenic-promoting potency of each of the above conditions in terms of cell morphology/viability, antigen presentation, and gene expression. DAG induced collagen I secretion from MSCs, which was further increased by the combination of DAG þ BMP-2. In comparison, the collagen scaffold and the control samples showed no significant influence on collagen I secretion of MSCs. DAG stimulation of MSCs led also to a steady but not significant increase of BMP-2 level. A DAG and more, a DAG þ BMP-2, stimulation increased the number of mesenchymal cells (CD105þ/CD73þ). All samples showed mRNA of ALP, osteopontin, Runx2, Twist 1 and 2, Notch-1/2, osteonectin, osteocalcin, BSP, and collagen-A1 after 28 days of in vitro culture. Culture media of all samples showed a decrease in Ca 2þ and PO 4 2À concentration, whereas a collagen-I-peak only occurred at day 28 in DAG-and DAG þ BMP-2-stimulated bone marrow cells. In conclusion, BMP-2 enhances DAG-induced osteogenic differentiation in mesenchymal bone marrow cells. Both agents interact in various ways and can modify osteoblastic bone formation. ß

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Section: Discussionsupporting

confidence: 91%

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Jäger

Fischer

Dohrn

et al. 2008

Journal Orthopaedic Research

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“…SDS PAGE stained with Coomassie Blue R250 revealed a single band with an apparent molecular size of 35 kDa that is fully consistent with our published studies [7]. In selected experiments LOX-PP expressed in bacteria was utilized [9].…”

Section: Recombinant Lysyl Oxidase Propeptidesupporting

confidence: 87%

“…The tumor suppressor activity of LOX has been mapped to the pro-peptide region [24] and LOX-PP inhibits tumor formation by breast cancer cells in mice [19]. In normal osteoblasts, LOX-PP accumulates in cultures [9] and recombinant LOX-PP is taken up by cells and accumulates over time associated with microtubules [7]. LOX is critically important for development of a normal cardiovascular system [17].…”

Section: Discussionmentioning

confidence: 99%

“…Stable transfectants were generated by Lipofectamine 2000 mediated transfection of TREX 293 cells (Invitrogen), and selected with 5 μg/ml blasticidin and 150 μg/ml zeocin. Resistant cells were induced with 1 μg/ml doxycycline for 48 hours, and media containing LOX-PP was purified on a nickel affinity column (BioRad Bio-Scale Mini Profinity IMAC Cartridge) essentially as described [9], followed by further purification by ultrafiltration. Urea was removed by exhaustive dialysis against water or PBS.…”

Section: Recombinant Lysyl Oxidase Propeptidementioning

confidence: 99%

“…Micrographs are from (A) sham-, and (B) injured-, femoral arteries (bar = 10 μm; n = 4); (L, lumen; N, neointima). (C) Western blot of a cultured neonatal rat aorta smooth muscle cell extract assayed with the LOX-PP antibody showing pro-lysyl oxidase (50 kDa), glycosylated LOX-PP (35 kDa) [7], and non-glycosylated LOX-PP (20 kDa) [9].…”

Section: Supplementary Materialsmentioning

confidence: 99%

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Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Hurtado

Vora

Sume

et al. 2008

Biochemical and Biophysical Research Communications

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Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.Tumor necrosis factor-α (TNF-α) contributes to the development of atherosclerotic lesions [15]. Important among its many activities, TNF-α promotes the proliferation of smooth muscle cells (SMCs) [15] and it stimulates matrix metalloproteinase (MMP) production [6]. Thinning of the fibrous cap by MMPs is undesirable due to the release of sequestered factors that leads to thrombosis [15]. TNF-α regulation of MMP-9 is of particular importance in atherosclerotic lesion development and in fibrous cap thinning [3,20]. Thus, up-regulation of MMP-9 expression by TNF-α can promote development of atherosclerotic lesions by multiple mechanisms that include stimulation of SMC proliferation and MMP-9 production.

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Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts

et al. 2018

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Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

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A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures

Cited by 72 publications

References 53 publications

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts

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