Hector A. Lucero

Lysyl oxidase: an oxidative enzyme and effector of cell function

Abstract.Lysyl oxidase (LOX) oxidizes the side chain of peptidyl lysine converting specific lysine residues to residues of α-aminoadipic-δ-semialdehyde. This posttranslational chemical change permits the covalent crosslinking of the component chains of collagen and those of elastin, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix. Four LOX-like (LOXL) proteins with varying degrees of similarity to LOX have been described, constituting a family of related proteins. LOX is synthesized as a preproprotein which emerges from the cell as proLOX and then is processed to the active enzyme by proteolysis. In addition to elastin and collagen, LOX can oxidize lysine within a variety of cationic proteins, suggesting that its functions extend beyond its role in the stabilization of the extracellular matrix. Indeed, recent findings reveal that LOX and LOXL proteins markedly influence cell behavior including chemotactic responses, proliferation, and shifts between the normal and malignant phenotypes.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by ∼2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.

Control of megakaryocyte expansion and bone marrow fibrosis by lysyl oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1low mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1low mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.

Cardioprotective Effects of a Selective B2 Receptor Agonist of Bradykinin Post-Acute Myocardial Infarct

BACKGROUND The cardioprotective benefits of bradykinin are attributable to activation of its B(2) receptor (B(2)R)-mediated actions and abolished by B(2)R antagonists. The current experiments evaluated the cardioprotective potential of a potent, long-acting B(2)R-selective agonist peptide analogue of bradykinin, the compound NG291. METHODS We compared the extent of cardiac tissue damage and remodeling and expression pattern of selected genes in mice submitted to acute myocardial infarct (MI) and treated for 1 week with either NG291 [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin or with saline delivered via osmotic minipump. RESULTS Active treatment resulted in better ejection fraction (EF) 69 +/- 1% vs. 61 +/- 3.1% (P = 0.01), (vs. 85 +/- 1.3% in sham-operated controls), fractional shortening (FS) 38 +/- 4% vs. 32 +/- 8% (NS) (vs. 53 +/- 1.2 in sham-operated controls), and fewer markers of myocyte apoptosis (TUNEL-positive nuclei 4.9 +/- 1.1% vs. 9.7 +/- 0.03%, P = 0.03). Systolic blood pressure (SBP) at end point was normal at 110 +/- 4.2 in actively treated mice, but tended to be lower at 104 +/- 4.7 mm Hg in saline controls with decreased cardiac systolic capacity. Expression patterns of selected genes to factors related to tissue injury, inflammation, and metabolism (i.e., the B(1)R, B(2)R, endothelial nitric oxide synthase (eNOS), TNF-alpha, cardiomyopathy-associated 3 (Cmya3), and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4)) showed that acute MI induced significant upregulation of these genes, and active treatment prevented or attenuated this upregulation, whereas the B(2)R agonist itself produced no difference in the myocardium of sham-operated mice. CONCLUSIONS Treatment with a selective B(2)R agonist initiated at the time of induction of acute MI in mice had a beneficial effect on cardiac function, tissue remodeling, and inflammation-related tissue gene expression, which may explain its structural and functional benefits.

The Role of Calcium on the Activity of ERcalcistorin/Protein-disulfide Isomerase and the Significance of the C-terminal and Its Calcium Binding A COMPARISON WITH MAMMALIAN PROTEIN-DISULFIDE ISOMERASE

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI) shows a 55% identity with mammalian protein-disulfide isomerase (PDI) (Lucero, H. A., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112–23119) is a high capacity low affinity Ca2+-binding protein and behaves as a Ca2+ storage protein in the ER of a living cell (Lucero, H. A., Lebeche, D., and Kaminer, B. (1998) J. Biol. Chem. 273, 9857–9863). Here we show that recombinant ECaSt/PDI bound 26 mol of Ca2+/mol and a C-terminal truncated mutant bound 14 mol of Ca2+/mol, both with aK d of 2.8 mm in 50 mm KCl and 5.2 mm in 150 mm KCl. The percentage reduction in Ca2+ binding in the mutant corresponded with the percentage reduction of deleted pairs of acidic residues, postulated low affinity Ca2+-binding sites. 5 mm Ca2+ moderately increased the PDI activity of both ECaSt/PDI and the C-terminal truncated mutant on reduced RNase and insulin. Surprisingly, ECaSt/PDI in the absence of Ca2+prevented the spontaneous reactivation of reduced bovine pancreatic trypsin inhibitor. In the presence of 1–5 mmCa2+ (or 10 μm polylysine) ECaSt/PDI augmented the bovine pancreatic trypsin inhibitor reactivation rate. In contrast, the C-terminal truncated ECaSt/PDI augmented rBPTI reactivation in the absence of Ca2+ and 1–5 mmCa2+ further accelerated the reactivation rate, responses similar to those obtained with mammalian PDI.

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010.

Cell Signaling, Internalization, and Nuclear Localization of the Angiotensin Converting Enzyme in Smooth Muscle and Endothelial Cells

The angiotensin converting enzyme (ACE) catalyzes the extracellular formation of angiotensin II, and degradation of bradykinin, thus regulating blood pressure and renal handling of electrolytes. We have previously shown that exogenously added ACE elicited transcriptional regulation independent of its enzymatic activity. Because transcriptional regulation generates from protein-DNA interactions within the cell nucleus we have investigated the initial cellular response to exogenous ACE and the putative internalization of the enzyme in smooth muscle cells (SMC) and endothelial cells (EC). The following phenomena were observed when ACE was added to cells in culture: 1) it bound to SMC and EC with high affinity (Kd = 361.5 ± 60.5 pm) and with a low binding occupancy (Bmax = 335.0 ± 14.0 molecules/cell); 2) it triggered cellular signaling resulting in late activation of focal adhesion kinase and SHP2; 3) it modulated platelet-derived growth factor receptor-β signaling; 4) it was endocytosed by SMC and EC; and 5) it transited through the early endosome, partially occupied the late endosome and the lysosome, and was localized to the nuclei. The incorporation of ACE or a fragment of it into the nuclei reached saturation at 120 min, and was preceded by a lag time of 40 min. Internalized ACE was partially cleaved into small fragments. These results revealed that extracellular ACE modulated cell signaling properties, and that SMC and EC have a pathway for delivery of extracellular ACE to the nucleus, most likely involving cell surface receptor(s) and requiring transit through late endosome/lysosome compartments.

Role of a pro-sequence in the secretory pathway of prothyrotropin-releasing hormone

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31–52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31–52 sequence. The decreased steady state level of the mutant ΔPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of ΔPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as “sorting signal” in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

Activation of cellular chemotactic responses to chemokines coupled with oxidation of plasma membrane proteins by lysyl oxidase

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of cellular LOX activity by preincubation of vascular smooth muscle cells (VSMC) with β-aminopropionitrile (BAPN), the irreversible inhibitor of LOX activity, resulted in the marked suppression of the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. Plasma membranes purified from VSMC not previously exposed to BAPN contained a group of oxidized plasma membrane proteins, including the PDGF receptor, PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to BAPN-free cells, which had been previously exposed to BAPN, restored the profile of oxidized proteins towards that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells was significantly diminished when compared with cells in which oxidation by LOX was prevented by BAPN. The chemotactic responses of LOX knock-out mouse embryonic fibroblasts mirrored those obtained with VSMC treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.

Quantitative histological image analyses of reticulin fibers in a myelofibrotic mouse

Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing via analysis of hundreds of composite images to identify a patchy nature of RF throughout the BM during disease progression in a mouse model of myelofibrosis. To this end, initial conversion of silver stained BM color images into binary images identified two limitations: variable color, owing to polychromatic staining of reticulin fibers, and variable background in different sections of the same batch, limiting application of the color deconvolution method, and use of constant threshold, respectively. By blind coding image identities, to allow for threshold input (still within a narrow range), and using shape filtering to further eliminate background we were able to quantitate RF in myelofibrotic Gata-1low (experimental) and wild type (control) mice as a function of animal age. Color images spanning the whole femur BM were batch-analyzed using ImageJ software, aided by our two newly added macros. The results show heterogeneous RF density in different areas of the marrow of Gata-1low mice, with degrees of heterogeneity reduced upon aging. This method can be applied uniformly across laboratories in studies assessing RF remodeling induced by aging or other conditions in animal models.