Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.

Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.

C2-ceramide, a cell permeable analogue of ceramide [CER] markedly reduced mitochondrial membrane potential [MMP] in insulin-secreting INS cells, which was followed by a significant accumulation of cytochrome c [Cyt c] into the cytosolic compartment. In a manner akin to CER, exposure of these cells to interleukin-1beta [IL-1beta] also resulted in reduction in MMP and cytosolic accumulation of Cyt c. Further, long-term exposure of these cells to either CER [but not its inactive analogue] or IL-1beta caused a marked reduction in their metabolic viability. However, unlike IL-1beta, which increased nitric oxide [NO] release, CER-treatment of INS cells had no effects of CER on NO release were demonstrable. Together, these findings suggest that CER-induced mitochondrial effects may not be mediated via iNOS gene expression and NO production. CER also activated an okadaic acid -sensitive protein phosphatase [CAPP] in the purified mitochondrial fraction, suggesting that CAPP might represent one of the target proteins for CER in the beta cell mitochondria. Together, our findings suggest direct detrimental effects of CER on mitochondrial function in beta cells leading to their dysfunction and demise via apoptosis. Moreover, our findings provide evidence for a potential difference in the mechanisms underlying CER- and IL-1beta-induced mitochondrial defects and apoptotic demise of the effete beta cell.

Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0-120 microg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80-120 microg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 microg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 microg CSC/ml. Moreover, 80-120 microg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.