Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation - PubMed (original) (raw)

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Paola A Hurtado et al. Biochem Biophys Res Commun. 2008.

Abstract

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-alpha-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.

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Figures

Figure 1

Figure 1

LOX-PP inhibits growth of SMC in cultures. In A, primary neonatal rat aortic SMCs were grown as described in the presence of LOX-PP (10 μg/ml) or vehicle and the number of viable cells per culture determined on day 2 n = 3; *, p < 0.0001). In B, Rat aortic SMCs were cultured, LOX-PP or vehicle was added to cultures for 24 hours. Tritiated thymidine incorporation was then determined. Values shown are the averages of 3 independent cultures (*, p < 0.005; **, p < 0.05).

Figure 2

Figure 2

LOX-PP inhibits (A) TNF-α stimulation of MMP-9 mRNA levels, and (B and C) Erk1/2 activation. In A, primary neonatal rat aortic SMCs were treated with or without 20 ng/ml TNF-α in the presence and absence of LOX-PP. MMP-9 and GAPDH mRNA levels were determined by qPCR with Taqman reagents. Data are pooled from two different experiments with three independent cultures each, and are expressed as a per cent of TNF-α-stimulated MMP-9 mRNA levels +/− SE, normalized to GAPDH. *, p<0.05; **, p<0.004, n=3. Primary neonatal rat aortic SMCs were cultured for 24 h in the presence or absence of 10 μg/ml LOX-PP or vehicle. Cells were then stimulated with 20 ng/ml TNF-α or vehicle (PBS) for 15 minutes. Cells were extracted and aliquots subjected to Western blotting for phosphorylated, and total Erk1/2, respectively. Panel (B) shows densitometric quantitation of phosphorylated Erk 1 and Erk 2 levels normalized to total Erk 1 and 2. *, p<0.05, n=3. Panel (C) contains a representative Western blot.

Figure 3

Figure 3

LOX-PP directly inhibits MEK2 activity, and not Erk2. Recombinant active MEK2 ( 1 ng) was incubated with 6.7 μg of inactive Erk2 in the presence or absence of recombinant LOX-PP purified from a bacterial expression system [LOX-PP (b)] [9] or from a mammalian expression system [LOX-PP (m)] at 30o C for 15 min. This incubation is stage I (Stg. I). Aliquots of Stage I reactions were then assayed for the activity of Erk2 by measuring the phosphorylation of myelin basic protein in the presence of [γ32P]-ATP [Stage II reaction (Stg. II)]. Some reactions contained LOX-PP only in the Stage II reaction. Data are means +/− SD of triplicate assays, and are from a representative experiment done three times with similar results (*, p<0.05).

Figure 4

Figure 4

LOX-PP epitopes occur at elevated levels in guidewire-injured femoral arteries. Eleven week-old mice were subjected to guidewire-induced femoral artery injury or sham surgery as described [11, 25] and sacrificed after 14 days. Sections were subjected to immunohistochemistry with anti-LOX-PP antibody. Arrows mark positive signals in smooth muscle cells. 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].

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