Iduronic acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells

Abstract

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.

Figure 1. DS-epi1−/− AoSMCs have reduced epimerase activity.

A, western blot analysis of 20 µg proteins of total cell lysate. DS-epi1 is visible as a band of around 100 kDa as indicated by the arrow. Aspecific band detected both in the WT and in the DS-epi1−/− cell extracts is marked with a star. Equal loading is shown by Ponceau Red staining. B, epimerase activity expressed as percentage of WT. Specific epimerase activity was 237 dpm/h/mg for WT AoSMCs. Values are mean ± SEM of triplicates.

Figure 2. Medium and cell surface DS-epi1−/− CS/DS have reduced IdoA content and lack IdoA-blocks.

WT and DS-epi1−/− AoSMCs were labeled with [³⁵S] sulfate and PGs released into the medium were isolated on a DE52-column and subsequently analyzed by size exclusion on a Superose 6 column. A-D, WT open circles, DS-epi1−/− filled circles. Elution profiles of PGs from medium of passage 2 (A) and passage 7 (B) cells are shown. Fractions indicated by a continuous line were pooled and analysed on SDS-PAGE after degradation with heparitinase and chondroitinase ABC (Fig. 2A and 2B, insets). Low molecular weight fractions from (B), corresponding to decorin, were pooled as indicated by a dotted line and CS/DS was purified. The split products after degradation with chondroitinase B (C) and chondroitinase ACI+ACII (D) were separated on a Superdex Peptide column. Elution positions of di- and tetrasaccharides are indicated by arrows; free sulfate peaks, derived from 4-sulfatase contaminating the lyases’ preparations, are indicated by stars. Quantification of total IdoA residues from the chromatogram in (C) is shown in the inset. Proportions of IdoA blocks, highlighted in (D) with a continuous line, are shown in inset. E, flow cytometric analysis of IdoA-containing epitopes in WT (left panel) and DS-epi1−/− (right panel) after cell surface staining of non-permeabilized AoSMCs using the anti-DS antibody GD3A12; negative control (no GD3A12 antibody), filled profile. F, confocal immunofluorescence microscopy of IdoA-containing epitopes using the anti-DS antibody GD3A12.

Figure 3. Cell surface HS epitopes change upon functional ablation of DS-epi1.

FACS analysis of AoSMCs from WT and DS-epi1−/− mice using a set of anti-HS antibodies. A) mAb-10E4; B) mAb-HepSS-1; C) mAb-NAH46; D) mAb-JM403. The fluorescence distribution profile of WT cells is shown by empty area, whereas the one of DS-epi1−/− cells is shown by shaded area . The mean value of the fluorescence channel of negative controls (no primary antibody added) was 2. The results shown are from cells at 80–90% confluence. A second experiment carried out on cells at 20–30% confluence gave superimposable results.

Figure 4. DS-epi1−/− cells are larger and contain less focal adhesion kinases.

A, Representative phase contrast image of WT and DS-epi1−/− cells captured 24 h after seeding. B, Calculated cell area from images shown in (A). Data are mean ± SEM (50 total cells of each genotype analysed). **p<0.01. C, Cells were seeded for two hours and harvested, followed by immunoblotting analysis of focal adhesion kinases FAK and pFAK. D, densitometric analyses of immunoblots shown in C. E, FAK mRNA was quantified by qRT-PCR on WT and DS-epi1−/− cells at different times after seeding. The quantification was done in quadriplicates and the experiment was repeated twice with similar results. ***p<0.001, **p<0.05, *p<0.01.

Figure 5. Directional migration is impaired in DS-epi1−/− AoSMCs.

Scratch assay was performed (A) and the repopulated area calculated (B). C, migration of WT and DS-epi1−/− cells in the presence of 48 h-conditioned media. D, migration of WT and DS-epi1−/− cells on pre-deposited matrix. C and D: WT cells with WT-conditioned medium/deposited matrix (white bar), WT cells with DS-epi1−/− conditioned medium/deposited matrix (grey bar), DS-epi1−/− cells with WT-conditioned medium/deposited matrix (dotted bar), and DS-epi1−/− cells with DS-epi1−/− conditioned medium/deposited matrix (black bar). Data shown are mean ± SEM of triplicates. E, 10 randomly selected cells were individually tracked for 15 hours after scratching and their (F) speed and (G) the distance covered were measured. For each experiment cells with different genotypes were seeded in two chambers and one scratch was made. Three fields/scratch were recorded. Two experiments were made with similar results. H, WT and DS-epi1−/− AoSMCs display an altered cell morphology and cytoskeletal organization during migration. Actin filaments of WT and DS-epi1−/− cells migrating in a scratch assay were stained by phalloidin (green); scale bars 50 µm. *p<0.05 **p<0.01, ***p<0.001).

Figure 6. Focal adhesion sites are decreased in the DS-epi1−/− cell membrane attached to substratum.

Total internal reflection fluorescence (TIRF) images were taken after (A, left panel) vinculin/phalloidin or (B, left panel) FAK/phalloidin double staining. Scale bars = 10 µm. Overlapping sites were measured with Manders’ coefficients in randomly chosen 10 cells (A and B, right panels). The staining experiments were repeated three times with similar results. ***p<0.001.