Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space

Abstract

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

Keywords: collagen; extracellular matrix; hydroxylysine (Hyl); lysine; lysyl hydroxylase 2; post-translational modification (PTM); secretion.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

LH2 underwent glycosylation and was secreted.A, anti-FLAG Western blotting of total cell lysates or conditioned media isolated from H1299 cells that had been stably transfected with empty vector (Vec) or FLAG-tagged wild-type (WT) or mutant LH2 lacking the N-terminal signal peptide (ΔN). Actin was used as a loading control for the total cell lysates. B, anti-FLAG Western blotting of lysates from H1299 cells that had been stably transfected with FLAG-tagged wild-type (WT) or mutant LH2 lacking the N-terminal signal peptide (ΔN) after a 5-h incubation of the lysates with (NFG) or without (Mock) N-glycosidase F. As an additional control, Western blotting was performed on lysates that had not been subjected to a 5-h incubation (−). Actin was used as a loading control. Each result is representative of findings from two independent experiments.

FIGURE 2.

LH2 co-localized with extracellular collagen in a human lung adenocarcinoma.A, fluorescence micrograph of a human lung adenocarcinoma tissue specimen stained with antibodies against LH2 and collagen I. Nuclei were counterstained with DAPI. Scale bar, 100 μm. The inset shows higher magnification of a region in which LH2 co-localizes with collagen I. LH2 and collagen I signals are pseudocolored in the overlay images. Scale bar, 30 μm. Two human lung adenocarcinomas were stained, and the results shown are representative of findings from both tumor specimens. B, to illustrate overlap of LH2 and collagen I in a confocal micrograph of a human lung adenocarcinoma tissue specimen (left), LH2 (green) and collagen I (red) staining intensities were plotted against distance along a white line in the image (left). C, the fraction of LH2 that co-localizes with collagen I within single fields (circles) of two human lung adenocarcinomas as determined by Pearson's correlation coefficient.

FIGURE 3.

LH2 co-localized with extracellular collagen in tumors generated by subcutaneous injection of DFC1024 lung cancer cells into nude mice. Representative electron microscopic images of a DFC1024 tumor tissue section stained with gold particle-labeled IgG or anti-LH2 antibodies. Gold particles that localize on extracellular collagen fibers (A) and in the cytoplasm of tumor cells (B) are indicated (arrows). Dashes indicate location of nuclear membrane (B). Scale bar, 200 nm. The results shown are representative of a single tumor generated in 1 mouse.

FIGURE 4.

LH2 formed homodimers in the extracellular space.A, schema of reporters containing full-length LH2 fused to the N-terminal (G1) or C-terminal (G2) domains of Gaussia luciferase (hGluc). LH2, G1, and G2 are color-coded (the legend). B, luciferase activities were measured in total cell lysates of 293F cells transiently transfected with the indicated reporters or empty vector (Vec). Data are the means ± S.D.) of triplicate samples. C, luciferase activities were measured in total cell lysates of 293F cells transfected with the indicated reporters or empty vector (Vec). Full-length LH2 (WT). Mutant LH2 lacking the putative dimerization motif (DD). Data are the means ± S.D. of triplicate samples. D, luciferase activities were measured in total cell lysates (Cell lysates) or conditioned media (Media) isolated from 293F cells transiently transfected with the indicated reporters or empty vector (Vec). Data are the means ± S.D. of triplicate samples. E, luciferase activities were measured in total cell lysates (Cell lysates) or conditioned media (Media) isolated from H1299 cells transiently transfected with the indicated reporters or empty vector (Vec). Data are the means ± S.D. of triplicate samples. The results shown are representative of two independent experiments.

FIGURE 5.

LH2 depletion decreased HLCCs and increased LCCs.A, the bar graph illustrates the results of Q-PCR analysis of LH2 mRNA levels in MC cells stably transfected with vectors expressing LH2 shRNA (SH5) or scrambled control shRNA (SCR). The results are expressed as the means ± S.D. of triplicate samples. The mRNA levels of the indicated transfectants are relative to those of the control transfectants, which were set at 100%. The blots on the right show the results of Western blot analysis of the MC transfectants. Actin was used as the loading control. The results shown are representative of two independent experiments. B–D, quantification of total collagen cross-links (B), HLCCs, LCCs, and HLNL (C) and the ratio of HLCCs-to-LCCs (D) in the MC transfectants. Total collagen cross-links was the sum of HLCCs (DHLNL+Pyr) + LCCs (HHMD) + HLNL. The HLCC-to-LCC ratio was calculated as (DHLNL+Pyr)/HHMD. Data are the means ± S.D. of triplicate biological samples in a single experiment. p values, 2-tailed Student's t test.

FIGURE 6.

Extracellular LH2 was sufficient to rescue the collagen cross-link switch.A, SDS-PAGE of immobilized metal affinity chromatography/gel filtration-purified wild-type LH2 (WT) or enzymatically-inactive D689A mutant LH2 (DA) under reducing (R) and non-reducing (NR) conditions. The positions of the molecular weight markers (MW) are indicated. The results shown are representative of two independent experiments. B, Hyl levels by amino acid analysis of (IKG)3 reacted with wild-type LH2. Control reactions were performed in the absence of (IKG)3 (LH2) or LH2 ((IKG)3). Each result is from one sample in a single experiment. C, Western blot analysis of MC_LH2-SH5 cells transfected with control vector (Vec) or vectors expressing WT LH2 or inactive LH2 (DA) to reconstitute intracellular and extracellular LH2. Actin was used as the loading control. The results shown are representative of two independent experiments. D–F, quantification of total collagen cross-links (D), HLCCs, LCCs, and HLNL (E), and the ratio of HLCCs-to-LCCs (F). Total collagen cross-links was the sum of HLCCs (DHLNL+Pyr) + LCC (HHMD) + HLNL. For each assay, the bars on the left are from cells treated with or without (Con) recombinant LH2 protein (treatment), and the bars on the right are from cells stably transfected with empty (Vec) or LH2 expression vectors (transfection). The HLCC-to-LCC ratio was calculated as (DHLNL+Pyr)/HHMD. The data are the means ± S.D. from triplicate biological samples in a single experiment. p values, 2-tailed Student's t test.