The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells

Abstract

The human osteosarcoma-derived cell line, SAOS-2, exhibits many of the phenotypic characteristics of osteoblasts including the deposition of types I and V collagens in an extracellular matrix. Lesser amounts of collagen XI chains were also detected. The cell layer collagen contains hydroxylysyl pyridinoline cross-links but without the accompanying lysyl pyridinoline typical of human bone collagen. This indicates that the lysine residues at the two helical cross-linking loci are fully hydroxylated. The isoform of lysyl hydroxylase, LH1, known to be required for full hydroxylation at these sites, was shown to be highly expressed by SAOS-2 cells. Our findings provide insight on the mechanism of post-translational overmodification of lysine residues in collagen made by osteosarcoma tumors, and may be relevant for understanding a similar overmodification observed in osteoporotic bone.

Figure 1

Intermolecular cross-links in type I collagen Diagram showing the sites, type and ratio of pyridinoline cross-links in bone type I collagen. Pyridinolines stabilize type I collagen fibrils of human bone at both ends of the molecule. HP is more abundant at the C-telopeptide-helix cross-link site (HP/LP ratio, 6:1) and LP is more abundant at the N-telopeptide-helix site (HP/LP ratio, 2:1) [19].

Figure 2

Biochemical analysis of collagen A) Collagen types synthesized by the SAOS-2 cell line in culture The medium and cell layer on pepsin digestion showed types I and V collagen α-chains on SDS-PAGE. The α1(I) and α2(I) chains ran slightly slower than pepsin extracted control preparations from human bone, suggesting post-translational over-modification. Mass spectrometry identified the band between the α1(V) and α2(V) to be a degradation product of the α1(V) chain. Bands marked “β” are dimers of collagen chains. B) Identification of α1(XI) collagen chains by mass spectrometry SDS-PAGE (6%) of collagen extracted by 1M NaCl from the extracellular matrix of SAOS-2 cells (+DTT). Arrow indicates a band identified as a pro-form of the α1(XI) collagen chain. The sequence of a tryptic peptide from this band unequivocally identified by mass spectrometry is shown. Mass spectrometry also identified pro-forms of α1(V) and α2(V), as well as processed α1(I) and α2(I) from this gel. Bands marked “β” are dimers of collagen chains.

Figure 3

Pyridinoline cross-links in SAOS-2 cell layer collagen compared with human bone RP-HPLC analysis of pyridinoline cross-links in the cell layer collagen showed HP alone (upper panel) in contrast with the ratio of about 2:1 for HP:LP typical of adult human bone (middle panel). This result indicates that the triple-helical domain cross-linking lysines were fully hydroxylated in the SAOS-2 collagen, whereas in bone collagen they are only partially hydroxylated.

Figure 4

Expression of lysyl hydroxylase mRNA a) 6% polyacrylamide gels showing the ethidium bromide-stained RT-PCR (35 cycles) products of LH 1, LH2, G3PDH and COL1A1 from human skin, mature human osteoblasts, SAOS-2 cells, cultured human chondrosarcoma cells (CH1) and cultured skin fibroblasts. b, c) A similar RT-PCR (25 cycles) experiment was performed after labelling with dCT³²P to assess relative levels of LH1 message. For each tissue or cell culture, RNA was amplified and resolved on a 6% gel. Autoradiograghs were scanned to compare bands quantitatively. As shown in the bar graphs, regardless of the RNA chosen as reference (COL1A1 or LH2), LH1 mRNA was much higher (10 to 30 fold) in SAOS-2 cells than in osteoblasts, skin and cultured cells.

Figure 5

Co-RT-PCR survey of LH1 mRNA levels in human tissues LH1 and G3PDH RNAs were co-amplified from fetal human tissues, using dCT³²P labelling conditions. a) Products were separated by electrophoresis on 6% polyacrylamide gels, and detected by autoradiography. b) Values represent the LH1 mRNA levels normalized to G3PDH as internal standard, as determined by quantitative scans of each lane. Large differences of LH1 mRNA levels were seen between the tissues, with particularly high levels in cartilage and muscle. None, however, exhibited levels approaching that of the SAOS-2 cells.

Figure 6

a: Northern Blot analysis of LH1 mRNA in SAOS-2 cells and skin 5 μg of SAOS-2, human fetal skin and RCS-LTC chondrocyte cell line total RNA was resolved on a 1.2% agarose-formaldehyde gel, blotted to nitrocellulose membrane, probed with a ³²P-labelled LH1 cDNA, and detected by autoradiography. RNA kilobase markers are shown at right. b: Quantitation of LH1 mRNA in SAOS-2 cells and skin by RNAase protection. RNA was hybridized to a ³²P-labelled LH1 antisense probe, digested with nuclease to remove the non-homologous portion of the probe, resolved on a denaturing 6% acrylamide gel, and detected by autoradiography. Lane 1, Full-length probe prior to nuclease digestion. Lanes 2-7, Control LH1 RNA reactions containing 1000, 300, 100, 30, 10, and 3 pg LH1 RNA respectively. Lanes 9-11, Replicate reactions containing 10 μg SAOS-2 RNA. Lanes 14 and 15, Replicate reactions containing 10 μg human fetal skin RNA. Lane 8 and12, Probe only. Lanes 13 and 16 are empty.

Figure 7

Expression of LH1 protein in cultured SAOS-2 cells SDS-soluble protein from SAOS-2 cells (lanes 1) and cultured human skin fibroblasts (lanes 2, 3) were separated by SDS-PAGE, blotted to PVDF membrane, probed with an antibody to LH1, and detected with a chemiluminescence based secondary antibody assay. The background bands below 66 kDa are unidentified.

Figure 8

LH1 gene copy number in SAOS-2 cells and human liver cells A 6% polyacrylamide gel showing multiplex-PCR products of the LH1 gene (PLOD1) and type X collagen gene. The relative abundance of PLOD1 and type X collagen PCR products was similar in SAOS-2 DNA and in normal fetal liver DNA, indicating a normal LH1 copy number in SAOS-2 cells. Thus, the high levels of LH1 mRNA are due to elevated expression and/or stability of these products in SAOS-2 cells.