Lysyl Oxidase Propeptide Inhibits FGF-2-induced Signaling and Proliferation of Osteoblasts

Abstract

Pro-lysyl oxidase is secreted as a 50-kDa proenzyme and is then cleaved to a 30-kDa mature enzyme (lysyl oxidase (LOX)) and an 18-kDa propeptide (lysyl oxidase propeptide (LOX-PP)). The presence of LOX-PP in the cell layers of phenotypically normal osteoblast cultures led us to investigate the effects of LOX-PP on osteoblast differentiation. Data indicate that LOX-PP inhibits terminal mineralization in primary calvaria osteoblast cultures when added at early stages of differentiation, with no effects seen when present at later stages. LOX-PP was found to inhibit serum-and FGF-2-stimulated DNA synthesis and FGF-2-stimulated cell growth. Enzyme-linked immunosorbent assay and Western blot analyses show that LOX-PP inhibits FGF-2-induced ERK1/2 phosphorylation, signaling events that mediate the FGF-2-induced proliferative response. LOX-PP inhibits FGF-2-stimulated phosphorylation of FRS2␣ and FGF-2-stimulated DNA synthesis, even after inhibition of sulfation of heparan sulfate proteoglycans. These data point to a LOX-PP target at or near the level of fibroblast growth factor receptor binding or activation. Ligand binding assays on osteoblast cell layers with 125 I-FGF-2 demonstrate a concentration-dependent inhibition of FGF-2 binding to osteoblasts by LOX-PP. In vitro binding assays with recombinant fibroblast growth factor receptor protein revealed that LOX-PP inhibits FGF-2 binding in an uncompetitive manner. We propose a working model for the respective roles of LOX enzyme and LOX-PP in osteoblast phenotype development in which LOX-PP may act to inhibit the proliferative response possibly to allow cells to exit from the cell cycle and progress to the next stages of differentiation.The process of bone formation is highly coordinated and has been divided into three identifiable stages as follows: proliferation, matrix maturation, and mineralization (1). Tightly controlled expression and action of growth factors and extracellular matrix molecules act in an autocrine and paracrine manner to promote osteoblast development and differentiation (2-4). The fibroblast growth factors (FGFs) 2 are a family of structurally related proteins, with 23 members identified (5). The role of FGF-2 in bone formation is highlighted by genetic studies in which overexpression of FGF-2 induces abnormal long bone formation, although its knockdown inhibits bone formation and reduces bone mass (6, 7). Additionally, several mutations in FGF receptors have been implicated in the etiology of human skeletal dysplasias (5, 8). FGF-2 is a key mitogen for various types of bone cells, including precursor cells, bone marrow stromal cells, calvarial osteoblasts, and mature osteoblasts (9 -12). FGF-2 contributes to the expansion of the pool of cells during the proliferative phase (9, 11, 13). Its role in the later stages of matrix maturation and mineralization is, however, controversial, with both positive (14 -17) and negative effects described (18 -21). Similar to effects of other mitogenic growth factors (22-25), chronic in vitro exposure of oste...

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“…Also, studies have found that LOX can bind to, and inhibit the signaling of, growth factors, including TGF-b1 and Fibroblast Growth Factor (FGF-2), albeit in non-cardiac cells. 46,47 The detrimental effects of TGF inhibition on the stressed heart were demonstrated using a dominant negative TGF-receptor mouse model. 48 In response to pressure overload, mice with reduced TGF Figure 8 Lysyl oxidase (LOX) inhibition (B) attenuated volume overload (VO)-induced increases in collagen I and III, but did not alter elastin expression.…”

The Lysyl Oxidase Propeptide Interacts with the Receptor-Type Protein Tyrosine Phosphatase Kappa and Inhibits β-Catenin Transcriptional Activity in Lung Cancer Cells

“…Consistent with these findings, Bouez et al showed that LOX protein expression was absent from human basal and squamous cell carcinomas and that a reduction in LOX mRNA levels but not enzyme activity caused a more invasive phenotype in a HaCaT skin equivalent model (3). In H1299 lung cancer cells, ectopic LOX-PP expression potently inhibited signaling by the endogenous mutant NRAS gene and the ability of these cells to form invasive colonies in Matrigel (46), while in prostate cancer cells, the addition of recombinant LOX-PP (rLOX-PP) protein decreased RAS signaling (45). Similarly, LOX-PP reduced fibronectin-stimulated signaling and the migration of Her-2/neu-driven breast cancer cells (49) and Ras signaling and the ability of pancreatic and breast cancer cells to form tumors in xenograft models in nude mice (30,31,46).…”

“…LOX is synthesized as a 50-kDa inactive proenzyme (pro-LOX), which is N-and O-glycosylated within sites of the propeptide domain (45), secreted, and then cleaved to the functional C-terminal ϳ30-kDa enzyme and an ϳ18-kDa N-terminal lysyl oxidase propeptide (LOX-PP). Another major function of the LOX gene was discovered with the observation that its expression inhibited the transforming activity of the H-Ras oncogene in NIH 3T3 fibroblasts (6,22).…”

Lysyl oxidase in colorectal cancer

“…1). During biosynthesis, the propeptide region is subject to N-linked (65) and O-linked glycosylation (68). Glycosylation of the propeptide has been shown to be essential for LOX enzymatic activity, but it is not required for secretion of the proenzyme (23).…”

“…It has been shown that the LOX propep- tide achieves its tumor suppressor effects by reentering the cell following cleavage (24), although the precise mechanism by which this is achieved remains unknown. Indeed, the propeptide contains a putative nuclear localization sequence (68), which suggests that it may be actively directed to the nucleus once released from the proenzyme, and it has been shown to be capable of both repressing the oncogenic bcl-2 gene in breast cancer (71) and inhibiting FGF-2 signaling in prostate cancer (53). It has also been shown to possess an ability to inhibit signaling pathways that lead to the activation of nuclear factor-B (NF-B) in prostate and lung cancer (51,52,62), suggesting that there are likely multiple mechanisms by which it may exert tumor suppressor activity.…”