Localization and activity of lysyl oxidase within nuclei of fibrogenic cells

Abstract

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Figure 1

Detection of intracellular proteins reactive with anti-LO by laser scanning confocal microscopy. (A) NRASMCs at ×400 magnification. (B) NRASMCs at ×1000 magnification. (C) 3T3 fibroblasts at ×400 magnification. (D) NRASMCs, preimmune (control) serum, at ×400 magnification.

Figure 2

(A) Detection of nuclear LO proteins by Western blot analysis. Lanes: 1, purified bovine aorta LO; 2, crude extract (16 mM potassium phosphate/6 M urea, pH 7.8) of bovine aorta; 3, extract of NRASMC nuclei; 4, NRASMC cytosol. (B) Western blots of cytosol fractions (lanes 1, 3, and 5) and nuclear extracts (lanes 2, 4, and 6) of NRASMCs probed with monoclonal antibodies to cytosol proteins. Probes used were as follows. Lanes: 1 and 2, anti-actin; 3 and 4, anti-calreticulin; 5 and 6, anti-Golgi-specific protein (58 kDa).

Figure 3

Urea gel electrophoresis-Western blot of purified 32-kDa bovine aorta and of extract of NRASMC nuclei probed with anti-LO. Lanes: 1, bovine aorta LO in 16 mM potassium phosphate (pH 7.8); 2, nuclei extracted into Nonidet P-40 buffer; 3, bovine aorta LO mixed with Nonidet P-40 extract of NRASMC nuclei before electrophoresis; 4, nuclei extracted into Nonidet P-40 buffer.

Figure 4

Catalytic expression in vitro of endogenous enzyme–substrate complexes in NRASMC nuclei. Solid bars, extract of isolated nuclei; hatched bars, intact nuclei. Nuc, nuclei; Cyt, cytosol. Cells were incubated in culture in the absence (Nuc or Cyt) or presence (Nuc/BAPN or Cyt/BAPN) of 100 μM BAPN. Results are presented as the means (bar heights) ± SD (error bars) of triplicate assays.

Figure 5

DPBA cross-link analysis of NaB³H₄-reduced proteins of NRASMC nuclei and cytosol. (A) Nuclear protein. The arrow indicates the elution position of reduced LNL. (B) Solid line, the same sample mixed with the DPBA derivative of synthetic reduced LNL; dashed line, reduced desmosine and isodesmosine standards. (C) Cytosol.

Figure 6

DPBA cross-link analysis of NaB³H₄-reduced proteins of NRASMC nuclei isolated from cells cultured in the presence (dashed line) or absence (solid line) of BAPN. (Inset) Profile of fractions 55–70 presented at an expanded scale.