Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

Abstract

LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN (β-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

Introduction

Lysyl oxidase is best known for its ability to strengthen the extracellular protein matrix, primarily through intermolecular crosslinking of collagen, the most abundant protein in higher organisms. LOX, the first lysyl oxidase isotype to be isolated, is exported as an inactive 50 kDa precursor and then cleaved to a 32 kDa active form (reviewed in [1]). In early work, β-aminopropionitrile (BAPN), the specific lysyl oxidase inhibitor [2], was found to cause extreme fragility of chick embryo connective tissues, an effect that could be quantified using the easy detachability of the embryo heads [3]. Not surprisingly, LOX^−/− mice die perinatally [4]. Other lysyl oxidase isotypes have been identified, such as LOXL1, which has activity similar to LOX [1].

The small GTPase Ras functions as a switch to activate or shut down cell proliferation. Mutant forms of Ras which are, in effect, stuck in the “on” configuration are found in about 30% of human cancers [5]. In NIH 3T3 fibroblasts, a downregulated gene was found in Ras-transformed cells that was re-expressed when interferon-treated cells reverted to normal phenotype; this “Ras rescission gene” was later identified as the gene encoding LOX [6]. Ectopic expression of LOX in Ras-transformed NIH 3T3 fibroblasts resulted in reduced colony formation and deactivation of NF-κB [7]. Downregulation of LOX by antisense transfection of normal rat kidney fibroblasts induced higher levels of active Ras, increased tumorigenicity, and upregulated cyclin D1 and the Wnt pathway effector β-catenin [8].

There is no consensus concerning the mechanism of action of LOX that results in its antioncogenic effect; in particular, the identity of the LOX binding partner(s) and/or substrate(s) that are involved. Di Donato et al. [9] found that microinjected LOX prevented Ras-induced Xenopus oocyte maturation, which required LOX activity. In contrast, LOX-induced reversion to normal phenotype of Ras-transformed NIH 3T3 fibroblasts reportedly required LOX 18 kDa propeptide only, with no requirement for enzyme activity [10]. Another study concluded that the entire LOX proenzyme was required for reversion from the transformed phenotype [11].

Cell proliferation, transformation, and differentiation are influenced by the identity of the extracellular matrix (ECM) proteins in contact with membrane-spanning integrins, by the number of contacts, and by the degree of rigidity of the ECM (reviewed in [12], [13], [14]). Lysyl oxidase dramatically affects the solubility and proteolytic resistance of its ECM protein substrates, and also the rigidity and tensile strength of the ECM, by intermolecular crosslinking. Consequently, experiments involving inhibition, silencing, downregulation, or knockout of lysyl oxidase may produce results due to changes in the nature, quantity, and/or physical properties of fibrous proteins in the ECM, or to altered actions of lysyl oxidase on other as yet unidentified proteins, or to a combination of these effects. Interpretation of such results would be simplified by experiments done with a cell line which does not synthesize and export ECM proteins that are known lysyl oxidase substrates. Evidence for the presence of intracellular LOX in neurons [15] suggests the possible presence of lysyl oxidase in PC12 cells, which do not manufacture the most abundant ECM protein, type I collagen.

Given that the LOX anti-Ras effect in oocytes occurs downstream of ERK2 in the MAPK pathway [9], and ERK2 translocates from the cytoplasm to the nucleus when activated, antioncogenic LOX may be located in cell nuclei. Previous work showed LOX immunoactivity and enzyme activity in the nuclei of smooth muscle cells and Swiss 3T3 fibroblasts [16], suggesting the possibility of LOX-induced nuclear protein crosslinking. In the present work, nuclear protein from cells grown with and without BAPN was assessed for changes in relative amounts of soluble and insoluble (possibly crosslinked) protein, leading to unexpected results. To the authors’ knowledge, this is the first report of a protein or enzyme activity required for limiting nuclear mass.