Keratocyte Phenotype Mediates Proteoglycan Structure

Cell Culture

Primary keratocytes were obtained from fresh bovine corneal stromae by collagenase digestion as described previously (35). The cells were diluted in serum-free Dulbecco’s modified Eagle’s/F12 medium containing antibiotics and cultured on tissue culture-treated plastic at 4 × 10⁴ cells/cm² (keratocytes) or 1 × 10⁴/cm² (fibroblasts and myofibroblasts) in a humidified atmosphere containing 5% CO₂. Culture medium was changed after 24 h (day 1) to Dulbecco’s modified Eagle’s/F12 medium with antibiotics (35) for keratocytes or the same containing 2% fetal bovine serum for fibroblasts and 2% fetal bovine serum with 2 ng/ml recombinant human TGF-β1 (Sigma-Aldrich) to induce myofibroblast formation. These media were changed at day 4, and cultures were harvested at day 5 or 6 as noted in the figure legends.

Glycosaminoglycans

Cultures were labeled with 100 μCi/ml carrier-free [³⁵S]sulfate (PerkinElmer Life Sciences) added on day 5 and the medium collected on day 6. In some experiments, 0.5 mM 4-nitrophenyl-β-D-xylopyranoside (N2132, Sigma-Aldrich) was added 1 h before labeling. Proteoglycans in the culture medium were purified by ion-exchange chromatography, dialyzed against water, and lyophilized. For chondroitin/dermatan sulfate quantitation, glycosaminoglycans from six identical 9.5-cm² cultures were dissolved and combined to make triplicate samples of 100 μl in 0.1 M Tris acetate, pH 8. These samples were digested with 2 μl of chondroitinase ABC (C3667, Sigma-Aldrich) at 10 units/ml, for 2 h at 37 °C. Digested products were recovered by ultra-filtration with Microcon YM-3 microfiltration devices (Millipore). Keratan sulfate was digested in a similar manner using a mixture of 0.2 milliunits of Escherichia freundii endo-β-galactosidase and 0.2 milliunits of keratanase II (Seikagaku) in 0.05 M sodium acetate, pH 6.5, at 37 °C overnight. The amount of labeled fragments liberated by digestion was determined by scintillation counting, corrected for non-digested controls, and normalized to protein content of the cells as described below.

For size determination, chondroitin/dermatan sulfate proteoglycans were separated from total ³⁵S-labeled proteoglycans by selective alcohol precipitation without enzymatic digestion of keratan sulfate (37). Protein was hydrolyzed with 20 μg/ml proteinase K twice for 30 min at 45 °C in 0.1 M Tris-HCl, pH 7.4, containing 0.1% SDS. Chondroitin/dermatan sulfate from xyloside-treated cultures were analyzed in the same manner. Keratan sulfate chains were obtained from total [³⁵S]proteoglycans by treatment with chondroitinase ABC (as above), dialysis, lyophilization, and proteinase K digestion in a similar manner. The protein-free [³⁵S]glycosaminoglycan chains were subjected to SDS-PAGE on 4–20% gels (chondroitin/dermatan sulfate) or 10–20% gels (keratan sulfate), electrotransferred in buffer without methanol to Genescreen Plus-charged nylon membranes (PerkinElmer Life Sciences), and subjected to autoradiography as described previously (35).

Fluorophore-assisted Carbohydrate Electrophoresis (FACE) Analysis of Glycosaminoglycans

Non-labeled proteoglycans were purified from media of triplicate 75-cm² cultures conditioned on days 4–6 as described above. They were digested with chondroitinase ABC in 0.1 M ammonium acetate, pH 7.5, or with combined keratanase II and endo-β-galactosidase in 0.1 M ammonium acetate, pH 6.5, followed by collection of the products by ultrafiltration as described above. Dried aliquots of the digestion products were fluorescently labeled with 5 μl of 0.1 M 2-aminoacridone in 3:17 acetic acid:dimethyl sulfoxide for 15 min followed by 5 μl of freshly dissolved cyanoborohydride 1 M at 37° overnight (38). Borohydride was quenched with 30 μl of 25% glycerol and 2 μl of 1 mg/ml bromphenol blue. The derivitized digestion products were separated on 8 × 10 × 0.05-cm gels of 28.5% acrylamide, 0.76% bisacrylamide containing 0.1 M Tris-HCl, pH 8. The running buffer was 0.089 M Tris borate, 2 mM EDTA, pH 8.4, chilled to 4 °C. This formulation duplicates the separation previously described for gels from Glyco Corp. (38) that are no longer available. Electrophoresis was carried out on ice at 5 watts of constant power/gel. Fluorescent bands were immediately photographed using a 12-bit Bio-Rad FluorS Max imaging system, and quantification was accomplished with Bio-Rad Quantity One software. FACE bands generated by chondroitinase were identified by co-electrophoresis with purified standards of fragments from chondroitin sulfate and hyaluronan (Sigma-Aldrich). Monosaccharide standards for keratan sulfate analysis were purchased from Sigma. Disaccharide standards were produced by digestion of purified bovine corneal keratan sulfate (Seikagaku) with endo-β-galactosidase or keratanase II, purified by size exclusion chromatography on a Superdex-Peptide column (Amersham Biosciences), and identified by FACE analysis as described previously (38). Molecular mass of the disaccharide keratan sulfate standards was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (39).

Histology

Cellular morphology was observed after 2 days in cultures fixed in 100% methanol for 20 min and then stained with 1% crystal violet in 20% ethanol for 30 min followed by destaining in water. The cells were photographed by Bright field optics with a ×20 objective. For cytoskeletal analysis, cells after 2 days culture were fixed in a room temperature paraformaldehyde (35) and double-stained with Alexa-488 labeled phalloidin (Molecular Probes) and with anti-vinculin clone hVIN-1 (Sigma-Aldrich) followed by goat anti-mouse labeled with Alexa-546 (Molecular Probes) using procedures described previously (35). 6-day cultures were similarly fixed and stained for α-smooth muscle actin with anti-smooth muscle actin (clone asm-1, Sigma-Aldrich) followed by Alexa-546 goat anti-mouse antibody in a similar manner. Cytoskeletal photographs were acquired on a Bio-Rad laser-scanning confocal microscope using a ×60 oil objective.

Real-time Reverse Transcriptase-PCR

Cells were collected by centrifugation after scraping into cold saline, and RNA was isolated using RNeasy mini kit (Qiagen). RNA was treated with DNase I (Ambion) according to supplier’s protocol and then concentrated by alcohol precipitation in the presence of GlycoBlue (Ambion). RNA was quantified by fluorimetry using RiboGreen (Molecular Probes).

RNA (400 ng) was transcribed to cDNA in a 100-μl reaction containing 1× PCR II buffer (Roche Applied Science), 5 mM MgCl₂, 800 μM dNTP mixture (Roche Applied Science), 2.5 μM random hexamers (Invitrogen), 0.4 units of RNase inhibitor, and 125 units of SuperScript II reverse transcriptase (Invitrogen). PCR was carried out for 40 cycles of 15 min at 95° and 60 min at 60° after an initial incubation at 95° for 10 min in an ABI7700 thermocycler. Reaction volume was 50 μl containing 1× TaqMan Buffer A (Applied Biosystems), 5 mM MgCl₂, 300 μM each dNTP, 0.025 units/ml AmpliTaq Gold polymerase, and 5 μl of cDNA. Forward and reverse primers and fluorescent internal hybridization probes for each gene, as shown in Table I, were used at optimized concentrations. Sequences for these genes were obtained from Gen-Bank^™ with the exception of that of the extra domain A form of bovine fibronectin. This information was obtained by direct sequencing of reverse transcriptase-PCR amplification products obtained from myofibroblast cDNA using primers based on published flanking sequence data. The bovine extra domain A sequence thus obtained was deposited in GenBank^™ with accession number AY221633. Amplification efficiency for each of the primer pairs shown was determined to be >90%.

For each gene/cDNA combination, amplifications without reverse transcriptase were carried out as negative controls. Amplification of 18 S ribosomal RNA was carried out for each cDNA (in triplicate) for normalization of RNA content. Threshold cycle number (C_t) of amplification in each sample was determined by ABI software. Relative mRNA abundance was calculated as the C_t for amplification of a gene-specific cDNA minus the average C_t for 18 S expressed as a power of 2, i.e. 2^ΔCt. Three individual gene-specific values thus calculated were averaged to obtain mean ± S.E.

Immunoblotting

Proteoglycans from culture media collected at days 4 –6 were digested with chondroitinase ABC or keratanase II and endo-β-galactosidase as described above. Samples from the digests normalized for cell number (by total cell DNA content) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes subjected to immunoblotting as previously described. Keratan sulfate, biglycan, and keratocan were detected in proteoglycans pooled from 3–4 individual cultures. Biglycan was examined in chondroitinase digests and keratocan after digestion of keratan sulfate. Cell layers were lysed directly in 1% SDS sample buffer. Protein was determined using the Micro BCA assay (Pierce) and DNA by fluorimetry with PicoGreen (Molecular Probes). Equal amounts of protein were separated by SDS-PAGE, 10% gels for ALDH and α-smooth muscle actin and 4 –20% for keratan sulfate and fibronectin. Proteins were either stained with Coomassie Blue (40) or alternately electrotransferred to polyvinylidene difluoride membranes and subjected to immunodetection (35) with antibodies to cellular fibronectin (clone IST-9, Accurate Chemical), ALDH (41), antibody J36 against keratan sulfate (37), a peptide antibody to biglycan (42), or monoclonal antibody to α-smooth muscle actin (Clone 1A4, Sigma-Aldrich). Keratocan was detected using an antibody generated against a mixture of 10 synthetic peptides, each encoding a unique amino acid sequence of bovine keratocan linked to keyhole limpet hemocyanin carrier. This antiserum was prepared and affinity-purified as described for anti-lumican peptide antibodies (43).

Morphology of Corneal Phenotypes in Vitro

Primary bovine keratocytes isolated from fresh stroma by collagenase digestion and cultured in absence of serum exhibited a dendritic (stellate) morphology (Fig. 1A) with multiple extended processes interconnecting individual cells. Phalloidin staining (Fig. 1D) revealed filamentous actin in the cortical region and associated with the cell-cell contacts at the intersection of the cell processes. Vinculin staining was weak, diffuse, and mostly perinuclear in localization. When cells prepared in a similar manner were exposed to 2% fetal bovine serum for 2 days, the cells became larger and flattened with a reduction in processes (Fig. 1B). Many cells were polarized with pseudopodial extensions (arrows) indicating motility. In these cells, filamentous actin formed stress fibers traversing the cell body (Fig. 1E). Vinculin was focally localized at the terminus of the actin fibers as is typical for matrix-adherent fibroblasts. Keratocyte cultures exposed to both fetal bovine serum and TGF-β1 contained larger, less refractile cells with a polygonal appearance. Fewer obviously motile cells were observed (Fig. 1C). Filamentous actin fibers were thicker and fewer in number than in fibroblastic cells (Fig. 1F). Vinculin accumulation in focal adhesion was denser and larger than in fibroblasts. After 5 days of culture, numerous cells were observed in which actin fibers were stained with antibodies to α-smooth muscle actin (Fig. 1G). Cells in serum-free medium (keratocyte phenotype) or grown in the fibroblastic phenotype did not exhibit α-smooth muscle actin staining (data not shown).

Expression of Phenotypic Markers

Primary stromal cells in conditions similar to those in Fig. 1 exhibited differential expression of a number of marker molecules. α-Smooth muscle actin, cellular fibronectin, and biglycan are associated with myofibroblasts in vitro and in vivo. Immunoblotting showed a marked abundance of these three proteins in TGF-β-induced myofibroblasts compared with keratocyte and fibroblast cultures (Fig. 2, A–C). Accumulation of ALDH was recently reported to be a distinguishing feature of keratocytes in vivo (44). This protein described as a corneal crystallin represents one of the major soluble proteins in keratocytes but is reduced in fibroblasts populating healing wounds. ALDH was detected in all of the cultured bovine stromal cells, but its concentration was markedly elevated in cells maintained in the keratocyte morphology (Fig. 2D). The immunostained ALDH band corresponded to a major protein of ~54 kDa visualized by Coomassie Blue staining, prominent in keratocyte cell lysates but not apparent in lysates from fibroblasts and myofibroblasts (Fig. 2E).

Keratan sulfate glycosaminoglycan chains and keratocan, a SLRP core of corneal keratan sulfate proteoglycan, are extracellular products highly enriched in the corneal stroma. Immunoblotting using monoclonal antibody J36 to keratan sulfate revealed heterogeneous high molecular weight keratan sulfate in proteoglycans isolated from keratocyte culture medium (Fig. 2F). In fibroblasts, J36 epitopes were reduced in molecular size to a band of 50 –60 kDa. In myofibroblasts, the J36 keratan sulfate epitope was not detected. Keratan sulfate-linked proteins secreted by keratocytes also contained abundant keratocan in the proteoglycans isolated from quiescent cultures of keratocytes (Fig. 2G). Keratocan was decreased in fibroblasts and almost undetected in myofibroblast cultures.

Real-time quantitative reverse transcriptase-PCR analysis assays were designed to detect mRNA for the five proteins identified in Fig. 2. Relative abundance of the transcript pools for these five proteins (Table II) showed that the protein expression levels detected by Western blotting was consistent with differences in mRNA pools for these proteins. Pools for α-smooth muscle actin, biglycan, and cellular fibronectin were increased 12–39-fold in myofibroblasts compared with keratocytes. Fibroblasts, however, had little increase in these mRNAs compared with keratocytes. Keratocan transcripts were decreased 15-fold in fibroblasts and 50-fold in myofibroblasts as compared with keratocytes. Similarly, ALDH transcript abundance was 700- and 2000-fold lower in fibroblasts and myofibroblasts, respectively, compared with keratocytes. These assays link gene expression associated with in vivo cell phenotypes with the cell culture model.

Collagen Expression

Collagen type I represents the major fibrillar collagen of the cornea, but synthetic levels of collagen I are low in adult non-wounded corneas (45). Collagen III is a fibrillar cornea present in fetal and wounded cornea but only a very minor component of adult corneal stroma (45). We previously found that mRNA and protein for collagen I and III were up-regulated in myofibroblasts compared with keratocytes (35). Real-time PCR analysis of the mRNA pools for these collagens (Table II) confirmed these increases in myofibroblasts. These assays also showed that, unlike other myofibroblastic markers, mRNA pools for collagens are up-regulated in fibroblasts as well as myofibroblasts.

Glycosaminoglycan Biosynthesis by Corneal Cells

Proteoglycans were metabolically labeled for 18 h with [³⁵S]sulfate and isolated from culture media by ion-exchange chromatography. In initial experiments, greater than 95% of sulfated glycosaminoglycan isolated from the media of the cultures was determined to be keratan sulfate and chondroitin/dermatan sulfate (data not shown). Thus heparan sulfate does not constitute a significant fraction of the soluble glycosaminoglycan secreted by these cultures. Keratan sulfate in the labeled proteoglycans, determined by sensitivity to endo-β-galactosidase and keratanase II, was reduced by ~40% in fibroblasts and ~ 60% in myofibroblasts compared with keratocytes (Fig. 3A). Conversely, ³⁵S-labeled chondroitin/dermatan sulfate measured by sensitivity to chondroitinase ABC was increased 3–3.5-fold in fibroblasts and myofibroblasts compared with keratocytes. In the presence of nitrophenyl-β-D-xyloside, a synthetic initiator of chondroitin polymerization, chondroitin/dermatan sulfate biosynthesis was increased >5-fold in all of the cultures as compared with cultures without this initiator (data not shown), suggesting that (as with many cell types) chain initiation represents a rate-limiting step in chondroitin and dermatan sulfate synthesis. In the presence of β-xyloside, fibroblasts continued to incorporate an approximate 3-fold more sulfate than keratocytes (Fig. 3C) but myofibroblasts increased the relative biosynthesis to almost 6-fold than that of keratocytes.

The size of the ³⁵S-labeled glycosaminoglycan chains was determined by polyacrylamide gel electrophoresis after proteolytic removal of the core proteins. Keratan sulfate produced by fibroblasts and myofibroblasts decreased compared with that of keratocytes, whereas chondroitin/dermatan sulfate chain length increased (Fig. 4, A and B). Chondroitin/dermatan sulfate made in the presence of β-xyloside was smaller than that without this initiator but did not increase in fibroblasts and myofibroblasts (Fig. 4C). These results suggest a relationship between rate of chain initiation and final chain length in chondroitin/dermatan sulfate.

Analysis of non-labeled chondroitin/dermatan sulfate secreted by the keratocyte cultures was carried out by FACE analysis after chondroitinase digestion. As shown in Fig. 5A, keratocyte cultures contained sulfated and non-sulfated disaccharides in approximately a 3:2 ratio. Sulfation was primarily on the 4 position of the N-acetylgalactosamine. In fibroblasts, the non-sulfated component was significantly lower and both 4-O- and 6-O-sulfation increased. In myofibroblasts, 4-O-sulfation represented the majority of the moieties and unsulfated chondroitin disaccharide was reduced to <5% of the total. Quantitation of chondroitin disaccharides is depicted in Fig. 5B. Hyaluronan was also detected in this analysis, and quantitation of hyaluronan secreted by the different cultures is shown in Fig. 5C. As shown, hyaluronan was not detected in keratocyte culture media but hyaluronan represented 1.5 and 4.5% of the chondroitinase-sensitive glycosaminoglycan in fibroblast and myofibroblast cultures.

A large number of fragments is generated by enzymatic depolymerization of keratan sulfate (46 –48). Characterization of these fragments has employed a variety of analytical approaches including FACE, a technique that can be used to quantitate major components of corneal keratan sulfate (38, 49). Digestion of keratan sulfate from keratocyte culture media with mixed keratanase II and endo-β-galactosidase generated eleven major bands visualized on FACE (Fig. 6A). Of these, monosaccharides and disaccharides involved in keratan sulfate chain extension constituted ~60% of fragments secreted by keratocyte cultures (Fig. 6B, black bars). The abundance of this set of fragments dropped ~5-fold in the media from fibroblast and myofibroblast cultures. The abundance of these chain extension fragments as a proportion of the total fragments was also reduced in the fibroblasts and myofibroblasts. Based on previous studies of keratan sulfate structure, it seems likely that most of the unidentified bands (Fig. 6B, gray bars) released by enzyme digestion represent moieties capping the non-reducing terminus of keratan sulfate. A variety of such capping structures has been documented in corneal keratan sulfate by NMR, and these components also are present in FACE analysis of keratan sulfate (38). These components showed no significant decrease in fibroblasts and myofibroblasts compared with keratocytes (Fig. 6B). Reduction of keratan sulfate chain length would reduce the ratio of chain extension moieties to capping fragments. Thus, the altered ratio of chain extension moieties to total degradation products in fibroblasts and myofibroblasts shown in Fig. 6B is consistent with a reduced keratan sulfate chain length as seen in Fig. 4.