Stimulation of Collagen Synthesis by Insulin and Proteoglycan Accumulation by Ascorbate in Bovine Keratocytes In Vitro

Abstract

purpose. Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma. The keratan sulfate proteoglycans (KSPGs) lumican and keratocan are also present, and they interact with collagen and modulate its assembly into fibrils. In this study, ascorbate was added to a defined medium containing insulin, and its effects on the synthesis of collagen and KSPGs by keratocytes were determined.

methods. Collagenase-isolated keratocytes were cultured with or without insulin with or without ascorbate. Collagen and glycosaminoglycan synthesis was determined by collagenase digestion of incorporated ³H-glycine and by chondroitinase ABC or endo-β-galactosidase digestion of incorporated ³⁵SO₄. KSPGs were detected by Western blot. Collagen stability was determined by pepsin digestion. Ethyl-3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation.

results. Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans. The accumulation of PGDS, a KSPG that does not interact with collagen, was not affected by ascorbate. Only the collagen synthesized in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation.

conclusions. The results of this study indicate that the accumulation of lumican and keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.

The corneal stroma contains keratocytes embedded in an extracellular matrix consisting primarily of collagen types I and V and of proteoglycans that contain either chondroitin sulfate (CS) or keratan sulfate (KS) chains. Electron microscopic studies show that the corneal stroma contains collagen fibrils of small, uniform diameter that are separated by small, uniformly sized spaces. The collagen fibrils in the corneal stroma are heterofibrils of collagen types I and V. ¹ Collagen type V is essential for the initiation of fibril formation, ² and the presence of collagen V in the heterofibril has been shown to limit the fibril diameter growth. ³ The proteoglycans are in the spaces between the fibrils in vivo, ⁴ and in vitro assays that measure collagen fibril assembly have shown that these proteoglycans modulate collagen fibril formation. ^{5 6 7} Collagen fibril formation in the presence of CS and KS proteoglycans purified from the cornea delay fibril formation, decrease the rate of fibril growth, and result in smaller collagen fibrils. ⁸ The removal of the GAG side chains did not affect the activity of the proteoglycans, but reduction and alkylation abolished the activity. This indicates that the core protein of these proteoglycans modulates collagen assembly into fibrils. ^{8 9} The major proteoglycans of the corneal stroma are decorin, ¹⁰ lumican, ^{11 12} and keratocan. ¹³ Decorin is a CS proteoglycan, whereas keratocan and lumican are KS proteoglycans. Keratocan-null ^{14 15} and lumican-null ^{16 17 18} mice have thinner corneas, and the collagen fibrils in the stromas are larger and less organized than in the stromas of normal mice, confirming the in vitro turbidimetry analysis of collagen fibril assembly. Taken together, these findings indicate that collagen type I assembly into fibrils is modulated by both collagen type V and the keratan sulfate proteoglycans in the stroma. 

Three procollagen polypeptides come together to form a left-handed triple helix immediately after synthesis. ^{19 20} Stable triple helix formation, however, can occur only if certain lysine and proline residues in the collagen molecule are hydroxylated. ²¹ This posttranslational hydroxylation is performed by either lysyl or prolyl hydroxylases. ²² These enzymes are found in the lumen of the endoplasmic reticulum (ER) ²⁰ and require ascorbic acid as a cofactor. ²³ Ascorbate deficiency in cell culture does not affect collagen synthesis but affects fibril formation and the rate of collagen secretion. ^{24 25 26} The nonhydroxylated collagen molecules denature at a lower temperature and in less stringent environments than properly hydroxylated collagen fibrils. ²⁷ Systemic ascorbate deficiency leads to scurvy, and this deficiency affects wound healing. Scorbutic wounds are weaker ²⁸ than nonscorbutic wounds, are prone to reopen, and contain a mass of irregular, unorganized collagen fibrils. ²⁹  

The cornea and anterior segment of the eye contain high levels of ascorbate, ³⁰ and it is thought to function as an antioxidant and protect the cornea from reactive oxygen species that result from UV irradiation. ^{31 32} The levels of ascorbate in the aqueous humor and the cornea decrease after an alkali burn, ³³ resulting in the cornea’s becoming scorbutic. The cells in the stroma show characteristics of scorbutic tissue such as a sparse ER, suggesting that they are not very metabolically active. ³⁴ Topical ascorbate has been used successfully to treat corneal alkali burns and has been proposed to do so by increasing the synthesis and secretion of properly folded collagen to replace the collagen denatured by the burn. ³⁵ In addition, topical ascorbate has been used after photorefractive keratectomy (PRK), and its use decreased the late onset of corneal haze. ³⁶ Ascorbate concentration is highest in the epithelium, the layer that is removed before PRK. Although the exact reason for the late onset corneal haze is not known, it is possible that removal of part of the epithelium before PRK results in a decrease in ascorbate levels in the anterior corneal stroma, and we further speculate that this decline may reduce the secretion of properly folded collagen, which could result in corneal haze. 

Ascorbic acid has been used to study collagen synthesis in culture, ²² but it is easily oxidized in solution and its metabolic by-products are cytotoxic in extended cell culture. ³⁷ A stable, nontoxic phosphate derivative of ascorbic acid (2-phospho-L-ascorbic acid) was developed ³⁸ and has been shown to stimulate collagen accumulation by skin fibroblasts in culture and also to enhance the secretion of type I and III collagen peptides by rabbit keratocytes cultured in medium containing fetal bovine serum. ³⁹ Serum, however, contains mitogens and morphogens that cause keratocytes in culture to proliferate, acquire a fibroblastic morphology, and cease keratocan expression. ^{40 41 42} A chemically defined medium containing insulin has been shown to stimulate keratocyte proliferation while maintaining the dendritic morphology as well as keratocan expression. ⁴³ In this study, keratocytes were cultured in this defined medium containing 2-phospho-l-ascorbic acid, to determine its effects on the synthesis and accumulation of collagen and KS proteoglycans. 

Materials and Methods

Chemicals

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Isotopes were obtained from PerkinElmer (Boston, MA). Gels, reagents, and equipment purchased from Invitrogen (Carlsbad, CA) were used to separate proteins and for transfer onto nitrocellulose. 

Cell Culture

Keratocytes were isolated from adult bovine corneas by using two sequential collagenase digestions as described elsewhere. ⁴⁴ Cells were plated in DMEM/F12 at high density (20,000 cells/cm²) in six-well plates (Corning-Costar, Cambridge, MA) and allowed to attach overnight at 37°C in 5% CO₂. The medium was changed the next day (day 1) and on days 4, 7, and 10 to fresh DMEM or DMEM supplemented with insulin (10 μg/mL), with or without 1 mM 2-phospho-l-ascorbic acid (ascorbate). Ethyl-3,4-dihydroxybenzoate (EDB) was added at 0.08- or 0.4-mM final concentrations on day 1. 

DNA Quantitation

Cell layers were harvested on days 1, 4, 7, and 10, to measure DNA content (Cyquant; Invitrogen). Briefly, the cell layers were rinsed with PBS, frozen, thawed, and solubilized in a lysis buffer supplemented with a DNA-binding dye. The DNA content was determined at 480/535 nm by measuring four wells in triplicate and comparing the values to a calf thymus DNA standard. The DNA content of parallel cultures was measured for experiments that used the cell layer. 

Cell Proliferation

Cultures were radiolabeled with 20 μCi [³H]thymidine/mL of medium for 72 hours beginning on days 1 and 4. The labeled medium was removed on harvesting and the cell layers washed with cold PBS. The cell layers were processed as described under DNA quantitation. Incorporation into DNA was determined as previously described. ⁴³  

Collagen Synthesis

The incorporation of ³H-glycine into collagen was determined as has been described. ^{45 46 47} In summary, cultures were incubated for 72 hours in medium containing 25 μCi ³H-glycine/mL beginning on day 1. The medium was adjusted to 4 M guanidine HCl and the cell layers were extracted in 4 M guanidine HCl. Unincorporated isotope in medium and cell layers was removed by chromatography on PD10 columns, equilibrated, and eluted with 4 M guanidine HCl (GE Healthcare, Piscataway, NJ). Fractions containing incorporated radioactivity were pooled and dialyzed against water, and 400-μL aliquots were incubated with or without 2.5 units of collagenase type III (Advance Biofactures, Lynbrook, NY) in collagenase digestion medium (3 mM N-ethyl maleimide, 50 mM Tris [pH 7.5], 150 mM NaCl, and 5 mM CaCl₂) for 3 hours at 37°C. Undigested proteins were precipitated with carrier BSA by using cold 10% TCA containing 0.5% tannic acid and released incorporated counts measured by liquid scintillation. The DNA content was determined as described earlier. 

Collagen Helix Stability

Medium from cells was adjusted to 0.5 M acetic acid and concentrated eightfold (Amicon Ultra spin concentrators, MWCO [molecular weight cutoff] 10,000; Millipore Corp., Bedford, MA). Fifty microliters of a 4-mg/mL pepsin solution (in 0.5 M acetic acid) per 3 mL was added, and the samples were rocked overnight at 4°C. Each sample received a second 50-μL aliquot from the pepsin stock, and digestion was allowed to continue for an additional 6 hours at 4°C. The samples were titrated with 1 N NaOH to pH 8.0, to inactivate the enzyme, dialyzed against water overnight, lyophilized, reconstituted in 1× SDS running buffer, and separated on 10% bis-tris gels in reducing conditions. The gels were stained according to the manufacturer’s protocol (SimplyBlue Safestain; Invitrogen). 

Proteoglycan Synthesis

Cultures were labeled with 50 μCi/mL of ³⁵SO₄ for 72 hours on day 1. The medium was collected, frozen, lyophilized, and reconstituted in 4 M guanidine HCl. Unincorporated isotope was removed with PD10 columns. Fractions containing incorporated ³⁵SO₄ were combined and concentrated using spin concentrators (Amicon; Millipore). Incorporation into CS and KS was determined by digestion with chondroitinase ABC or endo-β-galactosidase (Seikagaku Associates of Cape Cod, E. Falmouth, MA), as previously described. ⁴³ Aliquots containing equivalent incorporated radioactivity were fractionated (Superose 6 12/30 HR column; GE Healthcare, Piscataway, NJ), equilibrated, and eluted with 4 M guanidine HCl (0.05 NaAc [pH 6.5] at 0.3 mL/min. Fractions (0.6 mL) were collected and incorporation measured by liquid scintillation counting. The levels of CS and KS in each fraction were determined as described earlier. 

Western Blot Analysis

Core protein and protein levels in the media were measured by Western blot as previously described. ⁴³ In brief, the culture media were collected from each culture condition and concentrated by spin filtration to one tenth or one fiftieth of the original volume. The size of the aliquot needed from each sample to provide a similar signal on film by Western blot was empirically determined. The measured pixel density was then divided by the DNA content of the culture equivalent to the amount loaded and expressed as pixel density per microgram DNA. Blots were probed with polyclonal antibodies to bovine keratocan or lumican (diluted 1:1000) or prostaglandin D synthase (PGDS; 1:20,000). The lumican and keratocan antibodies are peptide antibodies raised against the core protein. ⁴⁸ The rabbit antiserum to bovine PGDS was a generous gift of Gary J. Killian (Penn State University). Membranes were rinsed and incubated for 1 hour in 1:10,000 horseradish peroxidase (HRP)-conjugated secondary IgG (GE Healthcare). Protein bands were visualized by chemiluminescence (ECL; GE Healthcare) on autoradiograph film (Bio-Max XAR; Eastman Kodak, Rochester, NY) and band density was measured (model GS-710 Calibrated Imaging Densitometer; Bio-Rad, Hercules, CA). 

Statistical Analysis

Statistical analysis was performed with Statview (SAS Institute, Cary, NC). Samples were analyzed by paired t-test. Standard error was used when n > 3 and SD when n = 3. 

Results

The DNA content of the keratocyte cultures was measured over a 10-day culture period, to determine whether ascorbate stimulates cell accumulation. The cells cultured in DMEM/F12 or DMEM/F12 medium supplemented with ascorbate did not increase in number, but keratocytes cultured in medium supplemented with insulin increased in number over the 10-day culture period (Fig. 1) . Cells cultured in insulin plus ascorbate showed a moderately higher initial accumulation rate than did keratocytes cultured in insulin in the absence of ascorbate and achieved the same density on day 7 as cells in insulin did on day 10. The incorporation of [³H]thymidine into DNA on days 1 to 4 and 4 to 7 reflected the cell accumulation rates in all 4 culture conditions (data not shown). 

The synthesis of collagen and proteoglycans was measured over 72 hours beginning on day 1 and ending on day 4, a period during which the cell number was rapidly increasing in the cultures receiving insulin. ³H-glycine incorporation into collagen was determined by sensitivity to collagenase digestion (Fig. 2) . Compared with the control, keratocytes cultured in ascorbate synthesized 40% less collagen (P < 0.05), and this decrease was in the collagen associated with the cell layer. Keratocytes cultured in insulin synthesized four times more collagen (P < 0.01), with increases of fivefold in the cell layer (P < 0.02) and threefold in the medium (P < 0.03). Cells cultured in both insulin and ascorbate synthesized seven times (P = 0.0004) more collagen than did the control—50% more than with insulin alone (P = 0.006). Compared with keratocytes cultured in insulin alone, the medium of keratocytes cultured in insulin plus ascorbate contained four times more collagen (P < 0.001), but the cell layer contained 30% less (P < 0.03). Keratocytes cultured in ascorbate plus insulin synthesized 11 times more collagen than did those cultured in ascorbate alone with a 9-fold increase (P < 0.001) in the cell layer and a 12-fold increase (P < 0.001) in the medium. These data suggest that insulin stimulates collagen synthesis and that ascorbate increases the proportion of the collagen that is secreted into the medium. 

The hydroxylation of proline in the procollagen molecule is a posttranslational modification necessary for the formation of a stable triple helix in the ER. Prolyl hydroxylases are the enzymes necessary for the hydroxylation of the 4 position of proline. Ethyl-3,4-dihydroxy-benzoate (EDB) is a selective inhibitor of prolyl hydroxylase. ⁴⁹ EDB is not toxic to the cells and has been previously shown to inhibit collagen deposition in a dose-dependent manner. ^{49 50 51} Because most of the collagen made by keratocytes cultured in ascorbate plus insulin was in the medium (Fig. 2) , the collagen in the medium was analyzed for stability by resistance to pepsin digestion. Keratocytes were cultured in insulin or insulin-plus-ascorbate-containing medium, with or without 0.4 mM EDB. The medium of the cells was collected, adjusted to 0.5 M acetic acid, digested with pepsin and analyzed by SDS-PAGE (Fig. 3) . The medium of keratocytes cultured in insulin plus ascorbate contained pepsin-resistant collagen type I and V fibrils, demonstrated by the prominent bands for α1(I) and α2(I), as well as a clear band for α1(V) between the 64- and 191-kDa markers (Fig. 3 , lane IA). The band for α2(V) partially comigrated with α1(I), but can be observed as a faint band just above α1(I) (Fig. 3 , inset; expanded view of lane IA). These bands were absent in the medium of cells cultured in insulin alone (lane I) and in the medium of cells cultured in insulin, ascorbate, and 0.4 mM EDB (lane IAE). These results show that the 2-phospho-l-ascorbic acid derivative of ascorbate acts on collagen to produce a stably folded triple helix. 

The accumulation of lumican and keratocan in the culture medium was determined by Western blot. The medium was digested with endo-β-galactosidase to remove the KS side chain before SDS-PAGE to facilitate transfer in Western blot. Antibodies to the core protein of the proteoglycan detect the core protein as a sharp band in a Western blot, ⁴³ and the pixel density of each band was determined (Fig. 4) . Treating cells with ascorbate alone increased keratocan and lumican levels in the medium fourfold (P < 0.005), but insulin alone had no effect on the levels of these proteoglycans. A ninefold increase in both lumican and keratocan was observed (P < 0.005 for both) when cells were cultured with insulin plus ascorbate. Prostaglandin D synthase (PGDS) is also synthesized as a KSPG by keratocytes in vitro. ⁴⁴ Consequently, we performed Western blot analysis for PGDS as well and found that neither ascorbate nor insulin affected PGDS levels. Because only keratocan and lumican have been shown to interact with collagen fibrils, ⁸ these results suggest that the increased accumulation of KSPGs in the medium is limited to those with core proteins that interact with collagen. 

Having shown that the accumulation of lumican and keratocan in the medium is increased by the addition of ascorbate, we then tested whether the stimulatory activity of insulin plus ascorbate could be inhibited by the addition of EDB at a low and high dose (0.08 and 0.4 mM, respectively). As previously shown, the addition of ascorbate to medium containing insulin increased lumican and keratocan accumulation, but the presence of 0.08 mM EDB in medium containing insulin and ascorbate reduced the accumulation of both lumican and keratocan by 19% and 28% (P < 0.01), respectively, after 72 hours (Fig. 5) . EDB at a high dose (0.4 mM) reduced lumican and keratocan (P = 0.07) accumulation in the medium to levels below those measured in insulin alone. The level of PGDS also declined, but the decrease was not significant (P = 0.5). The results of this experiment also confirm a correlation between hydroxylation and increased levels of lumican and keratocan production. 

Keratocyte cultures were radiolabeled with ³⁵SO₄, and the amount of incorporated radiolabel in the CS and KS secreted into the medium was determined by digestion with chondroitinase ABC and endo-β-galactosidase, to determine whether ascorbate would also increase the incorporation of ³⁵SO₄ into KS (Fig. 6) . Compared with the control, the addition of ascorbate alone did not increase the incorporation of ³⁵SO₄ into either CS or KS, and insulin caused only minor changes in the incorporation of ³⁵SO₄ into these glycosaminoglycans. Culture in insulin plus ascorbate, however, increased ³⁵SO₄ incorporation into both CS and KS significantly (P < 0.01), but preferentially enhanced incorporation into KS (11-fold for KS, 6-fold for CS), compared with control. 

Although most of the keratan sulfate made by keratocytes in culture has been shown to be in proteoglycans, a portion accumulates as free GAG in the medium. ⁴³ These GAG side chains may be the result of proteolytic degradation of the core proteins. Consequently, equivalent counts of incorporated ³⁵SO₄ present in the medium of insulin- or insulin plus ascorbate–treated cultures were fractionated (Superose 6; GE Healthcare) to separate intact proteoglycans from GAGs. The intact proteoglycans synthesized by keratocytes cultured in insulin or in insulin plus ascorbate eluted between fractions 15 and 23 (Fig. 7) . There was, however, relatively greater incorporation in fractions 19 to 23 for keratocytes cultured in insulin plus ascorbate. Fractions 15 to 23 were digested with chondroitinase ABC or endo-β-galactosidase to determine the glycosaminoglycan chain composition. The inset shows that >80% of the incorporated counts in fractions 15 to 18 were released by chondroitinase ABC digestion, whereas >60% of the incorporated counts in fractions 19 to 23 were released by endo-β-galactosidase digestion. This finding demonstrates that the KS-containing proteoglycans elute primarily in fractions 19 to 23 and further confirms that ascorbic acid preferentially stimulates the synthesis of KS-containing proteoglycans. 

Discussion

The results of this study indicate that ascorbate alone does not increase collagen synthesis by keratocytes in culture. The addition of insulin alone, however, increased collagen synthesis fourfold and the presence of ascorbate in medium containing insulin induced a further increase in collagen synthesis to sevenfold. The further increase in total collagen synthesis is probably due to the ascorbate-mediated hydroxylation of prolines and lysines in the collagen molecule that stabilizes the triple helix and thereby increases its secretion and resistance to degradation. Insulin has been shown to stimulate collagen type I synthesis in human lung fibroblasts at both the mRNA and protein levels, ⁵² and to stimulate collagenous protein accumulation in scleroderma fibroblasts through the PKC-γ pathway. ⁵³ Insulin therapy has been shown to improve wound healing of skin burns in rats. ⁵⁴ Insulin treatment has also been shown to improve the strength of healed through-and-through wounds in rabbit corneas. ⁵⁵ Because tissues derive their tensile strength from collagen, the increased strength of the healed corneal wounds treated with insulin may be due to increased collagen synthesis. 

The addition of ascorbate to culture medium containing insulin not only stimulated collagen synthesis, but also increased keratocan and lumican accumulation 9-fold and KS synthesis 11-fold. The increase in lumican and keratocan accumulation correlates with the increased synthesis of hydroxylated collagen. Culture in insulin alone stimulated collagen synthesis, but did not affect lumican and keratocan accumulation. Although insulin stimulated collagen synthesis in the absence of ascorbate the collagen did not form stable triple helices, as shown by a greater proportion of the total synthesized collagen accumulating with the cell layer or pericellularly ³⁴ and by the susceptibility of the collagen secreted into the medium to degradation by pepsin. When ascorbate was added to the insulin-containing medium, a higher proportion of the total synthesized collagen was secreted into the medium and the collagens type I and V present in the medium were pepsin resistant. The effect of ascorbate on stimulating lumican and keratocan accumulation and providing resistance of collagen to pepsin digestion was abolished when ethyl-3,4-dihydroxybenzoate (EDB) was added to the culture medium. EDB is an analogue of ascorbate and competitively inhibits prolyl hydroxylase, resulting in the synthesis of underhydroxylated collagen. ⁵¹ Taken together, these data suggest that the synthesis and accumulation of lumican and keratocan are linked to the level of collagen synthesis and to the stabilization of the collagen triple helix by hydroxylation. 

The ascorbic acid–mediated stimulation of keratan sulfate proteoglycan accumulated by keratocytes was limited to keratocan and lumican. Both keratocan and lumican have core proteins containing leucine rich repeats (LRRs). ^{56 57} Homology modeling of proteins with LRRs to RNase inhibitor show that these proteins fold into a solenoid tertiary structure ^{12 56 57 58} that interacts with the collagen fibril and regulates fibril formation and diameter. ⁸ PGDS is made as a keratan sulfate proteoglycan by keratocytes in culture, ⁴⁴ but it does not contain LRR and does not interact with collagen. Unlike keratocan and lumican, PGDS accumulation was not affected by insulin, ascorbate, or the combination of both. 

Our studies that show that while insulin stimulated collagen synthesis, most of this increase was in the collagen associated with the cell layer and that when ascorbic acid was included with insulin, most of the increase was in the collagen secreted into the medium, where the proteoglycans are also secreted. Proteoglycans such as lumican and keratocan that interact with collagen may depend on the formation of a stable collagen helix for their own stability. The core protein of these proteoglycans interact with specific regions of the collagen fibril ^{59 60 61} in a manner similar to the interaction of collagen with decorin. ⁶² The decreased stability of the collagen triple helix that forms in the absence of ascorbate would weaken if not abolish the interaction of the core protein with the collagen. The other possibility is that the core proteins for lumican and keratocan are also hydroxylated, and that it stabilizes their structure and prevents proteolytic attack. It may be interesting to note that elastin, another extracellular matrix component, is also hydroxylated in the presence of ascorbate. ⁶³ There is, however, no evidence that the prolines or lysines in lumican and keratocan are hydroxylated and the increased synthesis of these proteoglycans in the presence of ascorbic acid is more likely due to a protective effect that they receive by interaction with a stably folded collagen. 

Our study shows that ascorbate, besides stabilizing the collagen triple helix, also increases the accumulation of lumican and keratocan proteoglycans in the medium. These data suggest that the prophylactic action of ascorbate in alkali wound treatment ^{35 64} and in the prevention of late-onset corneal haze after PRK by the use of topical ascorbate, ³⁶ may be due to the effect of ascorbate on increasing the stability of the triple helix for collagen I and V which then acts to increase the stability and therefore the accumulation of the keratan sulfate proteoglycans lumican and keratocan. The increased accumulation of keratan sulfate proteoglycans, in turn, may accelerate the restoration of stromal transparency by regulating the assembly of the stably folded collagen triple helices into fibrils of the correct diameter. Insulin, which we and others ⁵³ have shown to stimulate collagen synthesis and which has been shown to improve wound healing, ^{54 55} may further enhance wound healing if used in combination with ascorbate, particularly in humans, since they lack the ability to synthesize ascorbate. 

 Supported by Grant EY08104 from the National Institute of Health (JRH).

 Submitted for publication June 6, 2006; revised August 8, 2006; accepted September 25, 2006.

 Disclosure: K. Musselmann, P; B. Kane, P; B. Alexandrou, P; J.R. Hassell, P

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Keratocyte population growth in culture using defined media. The addition of ascorbate to the culture medium increases initial growth rate when insulin is present in the medium but does not affect final cell density or growth in the absence of insulin (n = 4).

Figure 2.

 

Collagen synthesis and secretion in defined medium. Cultures were incubated in medium containing ³H-glycine for 72 hours. Radioactivity incorporated into collagen present in the medium and cell layer was determined with a collagenase specific for fibrillar regions of collagen. The addition of ascorbate alone did not alter the collagen synthesis. Adding insulin to the culture medium significantly increased collagen in both the cell layer and the medium. Insulin+ascorbate show a significant increase collagen secreted into the medium and a significant decrease in collagen in the cell layer compared with insulin alone (n = 3).

Figure 3.

 

SDS-PAGE analysis of medium digested with pepsin. The media of cells cultured in insulin (lane I); insulin+ascorbate (lane IA); or insulin, ascorbate, and EDB (0.4 mM; lane IAE) was collected, concentrated, and digested with pepsin in 0.5 M acetic acid. Aliquots of the digested samples and a pepsin-only control (lane P) were separated on 10% bis-tris and stained for total protein. Only the medium of cells cultured in insulin and ascorbate had pepsin-resistant collagen. Inset: expansion of lane IA, showing migration positions of the α chains for collagen I and V are labeled.

Figure 4.

 

Accumulation of keratan sulfate proteoglycans in the medium. Medium was collected on day 4, digested with endo-β-galactosidase, separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to keratocan, lumican, or PGDS. The Western blot analysis with antibodies against keratocan, lumican, and PGDS were scanned to determine their pixel density, and the net pixel density was divided by the DNA (micrograms) in each culture. Data are the mean of three determinations ± SD. Keratocan accumulation in medium containing ascorbate increased fourfold (P < 0.005) compared with the control. Medium containing insulin and ascorbate contained nine times more keratocan and lumican (P < 0.005) than control medium. The addition of ascorbate did not have an effect on PGDS levels, with or without insulin (n = 3).

Figure 5.

 

Accumulation of keratan sulfate proteoglycans in culture medium of cells treated with ethyl-3,4-dihydroxybenzoate (EDB). Keratocytes were cultured in medium containing 0.08 or 0.4 mM EDB in insulin-containing growth medium, with or without ascorbate. Samples were processed as described in Figure 6 . A dose-dependent significant decrease (P < 0.01) at 0.08 mM EDB was detected in both lumican and keratocan accumulation between cells cultured in insulin+ascorbate, and insulin+ascorbate+EDB. At 0.4 mM, lumican and keratocan levels were below the levels of the insulin-alone control.

Figure 6.

 

Incorporation of ³⁵SO₄ into glycosaminoglycans during 72 hours in culture. Cultures were incubated in medium containing ³⁵SO₄ for 72 hours. The culture medium was collected, and sensitivity of incorporated radioactivity to chondroitinase ABC and endo-β-galactosidase digestion was used to determine incorporation of ³⁵SO₄ into CS and KS, respectively. Bar, mean ± SD; n = 3.

Figure 7.

 

Chromatography of ³⁵SO₄ radiolabeled proteoglycans. Culture medium from keratocytes incubated with ³⁵SO₄ for 72-hours was harvested, and equal amounts of incorporated radioactivity was fractionated, to determine incorporation into intact proteoglycans. Inset: percentage of CS and KS in fractions 15 to 23 of medium of insulin+ascorbate–treated cells. Most of the incorporated radioactivity eluted at the position of the intact proteoglycans. Ascorbate preferentially stimulated the incorporation of ³⁵SO₄ into KSPG.