Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: Effects of TGF-β1 and serum deprivation

Gacheru, Stephen N.; Thomas, Kathleen M.; Murray, Stephen A.; Csiszár, Katalin; Smith-Mungo, Lynda I.; Kagan, Herbert M.

doi:10.1002/(sici)1097-4644(19970601)65:3<395::aid-jcb9>3.0.co;2-n

Cited by 71 publications

(45 citation statements)

References 43 publications

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“…Qualitatively, cultures that received 0.1 M CuSO 4 (but not 0.01 M) and HA fragments also contained far more elastin fibers than in controls (amorphous clumps only); LOX protein production was also 2.5 times as great and even more so (3.5 times) in the presence of HA fragments. The literature suggests strong interplay between transforming growth factor beta (TGF-b) availability and LOX, 51,52 which in turn may imply that Cu 2þ ion-induced increases in endogenous TGF-b release may have mediated the observed increases in LOX production. The increases in tropoelastin production in 0.1 M CuSO 4 -supplemented cultures could also be due to the TGF-b-mediated effect of Cu 2þ ions, although this hypothesis is subject to future validation.…”

Section: Discussionmentioning

confidence: 99%

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Kothapalli

Ramamurthi

2009

Tissue Engineering Part A

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Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo-and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu 2+ ) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu 2+ ions slowed cell proliferation (5.6 AE 2.3-fold increase over 21 days vs 22.9 AE 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 AE 0.4-fold), tropoelastin (4.1 AE 0.05-fold) and cross-linked matrix elastin (4.2 ± 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu 2+ ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu 2+ ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu 2+ ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers (*1 mm diameter). However, 0.01 M of Cu 2+ ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu 2+ ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.

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Section: Discussionmentioning

confidence: 99%

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Kothapalli

Ramamurthi

2009

Tissue Engineering Part A

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“…Analysis of over 13 kb of intervening sequence and 5Ј-flanking region of the human LO gene revealed a concentration of conserved consensus sequence elements within the first intron and 1-kb fragment immediately 5Ј of exon 1 (17). Human LO promoter activity in luciferase reporter constructs transfected into rat aortic smooth muscle cells was markedly elevated by serum deprivation (41). However, the detailed transcriptional regulation of the LO gene is still poorly understood.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

Gao

Zhao

Kong

et al. 2007

Journal of Biological Chemistry

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Lysyl oxidase (LO) stabilizes the extracellular matrix by crosslinking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from ؊78 to ؊51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5 Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region ؊709/؊598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment ؊612/؊580, which contains a nuclear factor I (NFI)-binding site at the region ؊594/؊580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at ؊147/؊133, were identified in the LO promoter region ؊804/ ؊1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation. Lysyl oxidase (LO)2 (EC 1.4.3.13) is a copper-dependent enzyme secreted by fibrogenic cells such as fibroblasts (1). This enzyme catalyzes the initiation of cross-linking of collagen and elastin, major structural components of the extracellular matrix (ECM), by oxidizing peptidyl lysine residues within these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde, leading to the formation of condensation products stabilizing polymeric collagen or elastin as insoluble fibers. Thus, LO plays a central role in ECM morphogenesis and tissue repair (1).In addition to the major function in stabilizing the ECM, LO also exhibits other biological activities. As reported, expression of transfected LO cDNA inhibited Ha-ras-induced cell transformation indicating an anti-tumorigenic effect of LO (2). LO can oxidize lysine residues in various globular proteins other than collagen and elastin (1). Oxidation of basic fibroblast growth factor (bFGF) by LO blocks the proliferation of bFGFstimulated cells and highly tumorigenic bFGF autocrine-transformed cells (3). Purified mature bovine LO displays chemotactic activity for monocytes and vascular smooth muscle cells (4, 5). LO and its oxidized substrates exist within the nuclei, potentially using histone H1 as a substrate and modulating the promoter activity of the collagen type III gene (6 -8). Increased LO activity is associated with fibrotic...

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“…In osteoblast cell cultures TGF␤ can inhibit collagenase 3 expression by accelerating the decay of its transcript (38). In vascular smooth muscle cells TGF␤ can stabilize lysyl oxidase mRNA (39). Other mechanisms of post-transcriptional regulation by TGF␤ have also been proposed.…”

Section: S]cys ϩ[mentioning

confidence: 99%

Sialomucin Complex (Rat Muc4) Is Regulated by Transforming Growth Factor β in Mammary Gland by a Novel Post-translational Mechanism

Price‐Schiavi

Zhu

Aquinin

et al. 2000

Journal of Biological Chemistry

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Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (for ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, produced from a single gene and precursor. SMC expression is tightly regulated in mammary gland; the level in lactating mammary gland is about 100-fold that in virgin gland. In rat mammary epithelial cells, SMC is post-transcriptionally regulated by Matrigel by inhibition of SMC precursor synthesis. SMC is also posttranscriptionally regulated by transforming growth factor-␤ (TGF␤). The repression of SMC expression by TGF␤ is rapid, is independent of TGF␤-induced cell cycle arrest, and does not require new protein synthesis. Unlike Matrigel, TGF␤ does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGF␤-treated and untreated cells. Instead, SMC precursor in TGF␤-treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGF␤ disrupts processing of SMC precursor. These results indicate that SMC, a product of normal mammary gland and milk, is regulated by TGF␤ by a novel post-translational mechanism. Thus, SMC is regulated by multiple posttranscriptional mechanisms, which serve to repress potential deleterious effects of overexpression.

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Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: Effects of TGF-β1 and serum deprivation

Cited by 71 publications

References 43 publications

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

Sialomucin Complex (Rat Muc4) Is Regulated by Transforming Growth Factor β in Mammary Gland by a Novel Post-translational Mechanism

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