Lysyl oxidase (lox) gene deficiency affects osteoblastic phenotype

Abstract

Lysyl oxidase (LOX) catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. The present study examined the role of Lox gene deficiency for the osteoblast phenotype in primary calvarial osteoblasts from E18.5 Lox knockout (Lox ( -/- )) and wild type (wt) (C57BL/6) mice. Next to Lox gene depletion, mRNA expression of Lox isoforms, LOXL1-4, was significantly downregulated in Lox ( -/- ) bone tissue. A significant decrease of DNA synthesis of Lox ( -/- ) osteoblasts compared to wt was found. Early stages of osteoblastic apoptosis studied by annexin-V binding as well as later stages of DNA fragmentation were not affected. However, mineral nodule formation and osteoblastic differentiation were markedly decreased, as revealed by significant downregulation of osteoblastic markers, type I collagen, bone sialoprotein, and Runx2/Cbfa1.

Figure 1

Lateral views of E 18.5 calcified tissues of Lox^-/- and wt mice stained with Alizarin Red and Alcian Blue. Experiments were performed three times. Representative images of one experiment are shown.

Figure 2

Three-dimensional reconstructions of of Lox^-/- and wt alveolar as well as of teeth-related structures. Reconstructions were performed two times for each genotype. Representative images (coronal (a-b), frontal (c) and dorsal (d) views) of one experiment are shown. Bone tissue is presented in brown, while Meckel's cartilage and teeth are given in blue and white colors, respectively.

Figure 2

Three-dimensional reconstructions of of Lox^-/- and wt alveolar as well as of teeth-related structures. Reconstructions were performed two times for each genotype. Representative images (coronal (a-b), frontal (c) and dorsal (d) views) of one experiment are shown. Bone tissue is presented in brown, while Meckel's cartilage and teeth are given in blue and white colors, respectively.

Figure 3

Electronmicroscopic analysis of collagen fibril diameters of craniofacial bone tissues. For each genotype, 500 fibril diameters were measured in randomly chosen areas. Representative images are shown for Lox^-/- and wt. (bar = 30 nm; magnification = × 35,000)

Figure 4

Real-time RT-PCR analysis of LOX and its isoforms, LOXL1-4, from Lox^-/- and wt mice. Data are presented as mean ± SD obtained from three measurents of pooled RNA samples. Asterisks indicate significant differences between Lox^-/- and wt mice (p<0.05; unpaired student's t-test assuming equal variances).

Figure 5

BrdU incorporation in primary Lox^-/- and wt osteoblastic cells after 24 h and 48 h measured by ELISA at 405 nm. Data are presented as means ± SD of three experiments (n = 6 cultures/genotype). Asterisks indicate significant differences between Lox^-/- and wt cells (p<0.05; unpaired student's t-test assuming equal variances).

Figure 6

Annexin-V positive (a), propidium iodine (PI) positive (b) cell count in primary Lox^-/- and wt osteoblastic cells after 6 h, 16 h and 24 h measured by fluorescence microscopy. Measurement of DNA fragmentation (c) in primary Lox^-/- and wt osteoblastic cells after 6 h, 16 h and 24 h by ELISA at 405 nm. Spectrophotometric measurement of LDH release (d) in primary Lox^-/- and wt osteoblastic cells after 6 h, 16 h and 24 h at 490 nm. Data are presented as means ± SD of three experiments (n = 3 cultures/genotype).

Figure 7

Real-time RT-PCR analysis of bone markers, COL1A1, bone sialoprotein (BSP) and Runx2/Cbfa1from Lox^-/- and wt mice. Data are presented as mean ± SD obtained from three measurents of pooled RNA samples. Asterisks indicate significant differences between Lox^-/- and wt cells (p<0.05; unpaired student's t-test assuming equal variances).