help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ganta, D. R.
Right arrow Articles by Gronowicz, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ganta, D. R.
Right arrow Articles by Gronowicz, G. A.
Endocrinology Vol. 138, No. 9 3606-3612
Copyright © 1997 by The Endocrine Society

ARTICLES

Ascorbic Acid Alters Collagen Integrins in Bone Culture1

Deepica R. Ganta, Mary-Beth McCarthy and Gloria A. Gronowicz

Department of Orthopaedics, University of Connecticut Health Center, Farmington, Connecticut 06032

Address all correspondence and requests for reprints to: Gloria Gronowicz, Department of Orthopaedics MC 1110, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: gronowicz{at}NSO1.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of ascorbic acid on collagen synthesis, mineralization, and integrins were investigated in a mineralizing organ culture system derived from 20-day fetal rat parietal bones. A significant dose-dependent decrease in calcification at 96 h was demonstrated with decreasing concentrations of ascorbic acid (100–0 µg/ml). No effect on DNA content, [3H]thymidine incorporation, or dry weight was found in control (100 µg/ml ascorbic acid) bones compared with bones treated with decreased ascorbic acid concentrations (10, 1, and 0 µg/ml). Collagen synthesis, measured by [3H]proline incorporation, and {alpha}1(I) procollagen messenger RNA levels were also unaffected. However, ascorbic acid produced a dose-dependent decrease in the hydroxyproline content, with a maximal 76.8% decrease in bones without ascorbic acid compared with the control bones with 100 µg/ml ascorbic acid. Light microscopy of the ascorbic acid-deficient bones revealed a disruption of the osteoblast layer with misshapen osteoblasts and a decrease in the osteoid seam. The loss of osteoblast organization was also confirmed by analyzing the integrins for collagen by Northern and Western blot and immunofluorescence microscopy. A dose-dependent decrease in {alpha}2 and ß1 integrin messenger RNA levels and in {alpha}1, {alpha}2, and ß1 protein were found in 96-h bone cultures deficient in ascorbic acid. These integrin subunits mediate the binding of osteoblasts to collagen. Immunofluorescence microscopy also demonstrated a dose-dependent decrease in {alpha}2 and ß1 staining of the osteoblast layer. However, the protein levels of {alpha}3 and {alpha}5 subunits were not affected. No ß5 was detected, whereas only bones cultured without ascorbic acid demonstrated a small decrease in {alpha}v and ß3 protein levels. The {alpha}3, {alpha}5, {alpha}v, and ß3 subunits are involved in cell binding to extracellular matrix proteins other than collagen. Thus, the integrins for collagen are down-regulated, probably in response to the underhydroxylated collagen fibrils, which causes a disruption of osteoblast organization leading to a decrease in mineralization of bone. Integrin assays for specific extracellular proteins may be useful tools in detecting matrix defects in various metabolic bone diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ASCORBIC acid is important for collagen synthesis in connective tissue due to its role as a cofactor for proline hydroxylase and lysine hydroxylase (1, 2), which are involved in the hydroxylation of collagen. A Na+-dependent transporter specific for ascorbic acid is present in the plasma membrane of osteoblasts and is essential for maintenance of intracellular ascorbate concentrations (3, 4). In limb bud rudiment bones from 7-day-old chicks, 50–100 µg/ml of ascorbic acid was necessary to produce a maximal increase in dry weight and collagen content in culture (5). Osteoblast proliferation (6, 7) and alkaline phosphatase expression (8, 9) were also dependent on ascorbate levels. Whether ascorbate’s effect on osteoblast growth and differentiation is mediated through collagen alone or through other pathways remains to be determined. One pathway, which was explored in this work, is the collagen receptors or integrins. In addition, osteoblast differentiation as mediated by 1,25(OH)2 vitamin D3, retinoic acid, and bone morphogenic proteins was also affected by the concentration of ascorbic acid (10, 11, 12, 13). Thus, ascorbic acid appears to be essential for normal bone formation.

Because integrins are able to transduce signals from the extracellular matrix to the intracellular compartment and to initiate proliferation and differentiation through various intracellular signaling pathways (14), their presence in osteoblasts may be important in regulating osteoblast function. Integrins have been shown to be involved in many cellular processes such as development, wound repair, tumor invasion, and inflammation (15, 16). Integrins are composed of an {alpha}ß heterodimer. Various combinations of the {alpha} and ß subunits produce receptors with different ligand specificities. There are 16 known {alpha} subunits and 8 known ß subunits (14, 15). The integrin superfamily has been shown to use primarily an arginine-glycine-asparate (RGD) sequence to recognize and bind ligands (15, 16). Thus, RGD peptides are competitive inhibitors of integrins that bind to these amino acid sequences in bone matrix proteins such as collagen, fibronectin, osteopontin, thrombospondin, vitronectin, and bone sialoprotein (17, 18). Puleo and Bizios (19) showed that the tetrapeptide RGDS inhibits the binding of primary rat osteoblasts to fibronectin (19). The intracellular domains of integrins are thought to interact directly with cytoskeletal proteins such as talin and {alpha}-actinin (20, 21, 22). In addition, the interaction of integrins with other intracellular proteins such as focal adhesion kinase, which binds the ß1 integrin cytoplasmic tail leading to the formation of focal adhesion sites (23, 24), initiates a cell signaling cascade that has been shown to regulate cell proliferation and differentiation (14).

The integrin subunits {alpha}v, {alpha}1, {alpha}2, {alpha}3, {alpha}4, {alpha}5, ß5, and ß1 but not ß2 have been found in human osteoblasts (25 28). The ß1 subunit, the major ß subunit in bone, is found in the receptors for collagen, fibronectin, laminin, and vitronectin. The ß3 subunit is also found in osteoblasts (25, 27, 28). Each integrin heterodimer mediates the binding of the osteoblast to a specific ligand or extracellular matrix protein found in bone. For collagen there are several possible integrin heterodimers for osteoblast adhesion to collagen; {alpha}1ß1, {alpha}2ß1, {alpha}3ß1, and {alpha}vß3. Binding to collagens I, IV, and V has been shown to involve different integrins (29); however, the specificity of the collagen receptors in osteoblasts is not known. Also, little is known about the regulation of osteoblast integrins by hormones or factors, such as ascorbic acid.

Previous work from our laboratory has shown that hormone and growth factors regulate the production of extracellular matrix proteins and their integrins. Glucocorticoids decrease the integrins for collagen and fibronectin in primary rat osteoblasts, transformed rat osteoblast cultures (30), and bone organ culture (31) and have been shown to have similar effects on collagen and fibronectin synthesis (31). Insulin-like growth factor I stimulated ß1 levels in bone organ culture (32) and is known to be anabolic for many of the bone matrix proteins and bone formation. Therefore, we undertook this study to determine whether a nonhormonal agent, such as ascorbic acid, would alter the collagen integrin along with collagen synthesis, or whether integrins could be independently regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture conditions
Fetal 20-day-old parietal bones were isolated from pregnant Sprague-Dawley rats (Charles River Farms, Wilmington, MA) and cultured as described previously (33) in Fitton-Jackson-modified BGJb medium (Gibco BRL, Grand Island, NY) with 0, 1, 10, or 100 µg/ml ascorbic acid on a rocking platform at 37 C in a 5% CO2 atmosphere. The medium was supplemented with 1% ITS+ containing 6.25 µg/ml transferrin, 6.25 ng/ml selenous acid and insulin, 1.25 mg/ml BSA, and 5.35 µg/ml linoleic acid (Collaborative Research, Lexington, MA). The medium was changed daily.

Dry weight and calcium content
Parietal bones were extracted twice for 30 min with 1 ml 5% trichloroacetic acid (TCA). Calcium content was measured in the pooled TCA washes by a colorimetric assay with o-cresolpthalein complexone (Sigma, St. Louis, MO). The decalcified bones were washed twice with 1 ml acetone and twice with 1 ml ether, air dried for 24 h, and weighed.

DNA content
The TCA extracted and dried bones were homogenized in 0.5 M acetic acid. The DNA content of the homogenate was measured according to the fluorimetric method of Kissane and Robbins (34).

Immunofluorescence
Bones were fixed with 5% paraformaldehyde and 2% sucrose in 0.1 M sodium cacodylate buffer, pH 7.4, for 1.5 h on ice, washed in 5% sucrose in buffer, and frozen in liquid nitrogen. Cryostat sections were cut and placed on chrome-alum-treated slides (0.5% Knox gelatin and 0.05% chromium potassium sulfate dodecahydrate). Sections were washed once in PBS, incubated with 1% gelatin in PBS, and treated with a 1:50 dilution of an affinity purified polyclonal rabbit antibody to the human integrin subunit ß1 (Chemicon International Inc., Temecula, CA) or a monoclonal antihuman {alpha}2 integrin antibody (Gibco BRL) for 2 h. After rinsing three times with PBS, the sections were incubated in a 1:300 dilution of rhodamine-conjugated goat antirabbit IgG (Chemicon) for 1 h. The tissue was visualized with a Nikon Optiphot fluorescence microscope (Nikon Co., Melville, NY), with 2.5% n-propyl gallate in 1:1 PBS/glycerol to prevent quenching.

Western blot analysis
Protein was isolated from bones by solubilizing the samples in RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, pH 7.4) containing the protease inhibitors 10 µg/ml aprotinin, 2 µg/ml pepstatin, and 2 µg/ml leupeptin (Boehringer Mannheim) and 0.5 mM phenylmethylsulfonyl fluoride (Sigma). The protein concentration was determined by using a BCA protein assay kit obtained from Pierce (Rockford, IL), and 70 µg of protein was boiled in sample buffer consisting of 2% SDS, 10% glycerol, 60 mM Tris, pH 8.8, and 0.001% bromophenol blue. The proteins were electrophoresed in SDS polyacrylamide gels and transferred to polyvinylidine difluoride (Millipore, Bedford, MA) according to the manufacturer’s directions. The membranes were blocked in T-TBS (0.1% Tween 20 in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl) with 5% skim milk powder. After washing in T-TBS, the blots were incubated with the appropriate antibody for 2 h at room temperature. Antibodies to {alpha}1, {alpha}2, {alpha}3, {alpha}5, {alpha}v, ß1, ß3, and ß5 were obtained from Chemicon. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Pierce), and positive bands were detected using the Pierce chemiluminescence kit. Blots were also stripped for reprobing with different primary antibodies by incubating in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.8, for 30 min at 50 C. Relative protein levels were determined by densitometry with the Scan Maker IIsp (Microteck Lab Inc., Redondo Beach, CA) and Sigma Scan (Jandell Co., San Rafael CA). The densitometry of two-thirds of each band (entire width and diameter) was measured. From this densitometric number, the background densitometry of the autoradiograph from the same region above each band was subtracted. Three scans were performed on each band, and the SE of the mean was determined. The densitometric scan of the integrin band from the bones treated with 100 µg/ml ascorbic acid was arbitrarily set at 100, and the other values for the integrin bands found in bones treated with varying concentrations of ascorbic acid were expressed relative to the 100 µg/ml ascorbic acid band. Western blots were performed for each integrin subunit three times with similar results.

Collagen synthesis
During the last 2 h of the culture period, 10 µCi/ml [3H]proline (44.5 Ci/mmol) (DuPont Co., Boston, MA) was added to each bone. After labeling, the bones were extracted and dried as described above and homogenized in 0.5 N NaOH. To determine the incorporation of [3H]proline into collagen, the bone homogenates were digested with purified bacterial collagenase according to the method of Peterkofsky and Diegelmann (35). The percentage of collagen being synthesized was corrected for the relative abundance of proline in collagen-digestible protein (CDP) and noncollagen proteins (NCP) (36). Bones were also labeled with 10 µCi/ml [3H]proline for 4 h at 20 h of culture. Tritiated proline was measured in the medium after each daily change of medium until 96 h to determine whether newly synthesized collagen was being released from the extracellular matrix due to degradation or changes in collagen fibril formation.

Thymidine incorporation
During the last 2 h of the culture period, 10 µCi/ml methyl[3H]thymidine (DuPont) was added to each bone. At the end of the culture period, bones were extracted, dried, homogenized in tissue solubilizer (Soluene, Packard, Meriden, CT), and counted in a scintillation counter.

Northern blot analysis
RNA was obtained from 10–12 parietal bones by using the thiocynate-phenol chloroform extraction method of Chomczynski and Sacchi (37). RNA was denatured, eletrophoresed through 0.8% agarose containing 2.2 M formaldehyde, and transferred to a nylon membrane in 10x SCC via Posiblot (Stratagene, La Jolla, CA). The bound RNA was immobilized in an UV Stratalinker (Stratagene), prehybridized, and then hybridized in 50% formamide, 5x SSC, 0.1% SDS, 0.1 M NaPO4, 5x Denhardt’s solution, and 100 µg salmon sperm DNA/ml at 42 C. The human ß1, {alpha}2, and {alpha}3 complementary DNA (cDNA) inserts were obtained from Telios Pharmaceuticals (Gibco BRL). The {alpha}1(I) procollagen cDNA probe was obtained from Dr. Barbara Kream (38). The actin probe was generously given to us by Dr. Don Cleveland. These probes were labeled with [32]deoxy-GTP using random primer nucleotides (39). The filter was washed with 2x SCC/1% SDS at 65 C and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -20 C. Relative hybridization levels were determined by densitometry. Each band was normalized to the actin band to correct for any loading discrepancies. The {alpha}2 and {alpha}3 human cDNA probe had difficulty hybridizing with the rat messenger RNA (mRNA), and the film required long exposure times. This may be due to poor homology in sequence between human and rat integrin subunits. The {alpha}1 and {alpha}5 human cDNA probes were unable to hybridize with rat RNA.

Statistical analyses
Data were analyzed using a nonparametric one-way ANOVA, followed by Student’s, Neuman-Keuls test to determine significance between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
During 96 h of culture, 20-day-old fetal parietal bones demonstrated significant growth. These control bones were cultured with 100 µg/ml ascorbic acid. The bones displayed a 2.0-fold increase in calcium content from 9 ± 1 µg to 18 ± 1 µg, and a 1.6-fold increase in dry weight from 137 ± 7 µg at 0 time to 216 ± 13 µg at 96 h. Decreasing the concentration of ascorbic acid to 10, 1, or 0 µg/ml had no significant effect on dry weight, DNA content, or thymidine incorporation at 96 h (Table 1Go). Ascorbic acid did not affect calcium content of the bones cultured for 24 h (Fig. 1Go). However, at 96 h, calcium content decreased by approximately 35% with 1 µg/ml and 0 µg/ml ascorbic acid in comparison with bones treated with 100 µg/ml ascorbic acid (Fig. 1Go) (P < 0.01).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of ascorbic acid on dry weight, DNA, and thymidine content in 20-day-old fetal rat parietal bones for 96 h

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Effect of varying concentrations of ascorbic acid on calcium content in 20-day-old fetal rat parietal bones. Bones cultured for 24 h with 0, 1, 10, and 100 µg/ml ascorbic acid showed no significant difference in calcium content. At 96 h, calcium content was significantly decreased with 0 and 1 µg/ml ascorbic acid compared with control bones treated with 100 µg/ml ascorbic acid. Three experiments with a total of 14–15 bones per group. Bars, Mean ± SEM; *, P < 0.01 compared with 100 µg/ml at 96 h.

 
Light microscopic examination of parietal bones cultured for 96 h with 100 µg/ml ascorbic acid showed a contiguous layer of osteoblasts along osteoid and a smoothly contoured mineralized matrix (Fig. 2AGo). Bones treated with lower concentrations of ascorbic acid, 10 µg/ml, 1 µg/ml, and 0 µg/ml ascorbic acid (Fig. 2BGo, C, and D, respectively) demonstrated misshapen and disorganized preosteoblasts and osteoblasts. In bones treated with 1 and 0 µg/ml ascorbic acid, the mineralized matrix was unevenly contoured, and the osteoid seam was narrow and discontinous compared with the control bones.



View larger version (183K):
[in this window]
[in a new window]
  		Figure 2. Light microscopy of bones treated for 96 h with 100 (A), 10 (B), 1 (C), and 0 (D) µg/ml ascorbic acid. Arrows in B, C, and D show osteoblasts, located along osteoid seam overlying mineralized matrix, which are swollen and discontiguous in comparison with osteoblasts in control bone (A). In addition, the mineralizing front is uneven in bones treated with 1 µg/ml (C) and 0 µg/ml (D) ascorbic acid. Magnification, x250; bar, 20 µm.

 
No significant differences in the percent collagen synthesized, as determined by [3H]proline incorporation into CDP or NCP, was found between groups treated with varying concentrations of ascorbic acid for 24 h (not shown) and 96 h (Table 2Go). No differences in CDP and NCP were apparent. For assaying [3H]proline release into the medium, bones were pulsed in the last 4 h of a 24-h period of culture with varying concentrations of ascorbic acid. New medium was added, and the bones were cultured for an additional 72 h for a total of 96 h with a daily change of medium. The amount of [3H]proline in the daily medium did not differ between groups (data not shown), suggesting that there was no increased release of collagen or its fragments into the medium.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of ascorbic acid on collagen synthesis in 20-day-old fetal rat parietal bones for 96 h

 
Northern analysis of {alpha}1(I) procollagen mRNA at 96 h was also unaffected by varying concentrations of ascorbic acid (Fig. 3Go, top lane). Therefore, both mRNA and protein levels of type I collagen were not changed. However, the hydroxyproline content of the bone was 76.8% less for bones cultured without ascorbic acid and 50.0% less for bones treated with 1 µg/ml ascorbic acid in comparison with the control group (100 µg/ml ascorbic acid) (Fig. 4Go). These results reveal that collagen synthesis continued at the same level, but the collagen produced was substantially underhydroxylated in bones without ascorbate.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. A, Northern blot of mRNA expression in bones treated with varying concentrations of ascorbic acid. No change in {alpha}1(I)pro-collagen mRNA was found. B, Changes in integrin levels seen in Western blots. Each band was compared by densitometry to ß-actin, which was used to normalize RNA levels. Density of each integrin band was plotted relative to density of control, 100 µg/ml ascorbic acid band( set arbitrarily at 100). A dose-dependent decrease in mRNA levels of {alpha}2 and ß1 subunits was observed, whereas no significant change in {alpha}3 integrin mRNA was found. Three experiments. Bars, Mean ± SEM.

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Hydroxyproline content of bones cultured for 24 and 96 h with 100, 10, 1, and 0 µg/ml ascorbic acid. Hydroxyproline content of bones treated with varying concentrations of ascorbic acid for 24 h was unchanged. Bones cultured for 96 h with 0, 1, and 10 µg/ml ascorbic acid showed a significant decrease in comparison to control bones treated with 100 µg/ml ascorbic acid. Two experiments with a total of 12 bones. Bars, Mean ± SEM; *, P < 0.01 compared with 0 µg/ml at 96 h.

 
Next, we studied the effect of ascorbic acid deficiency on the message levels of receptors for collagen on osteoblasts at 96 h. {alpha}2ß1 is a major collagen integrin in bone, but it can also bind laminin (15). Northern blot analysis demonstrated a dose-dependent decrease in {alpha}2 and ß1 mRNA levels (Fig. 3Go). There was a 74.6% and 96.4% decrease in {alpha}2 and ß1, respectively, in bones cultured without ascorbic acid. No effect on the {alpha}3 subunit was found. The integrin {alpha}3ß1 binds collagen, laminin, and fibronectin (15). Other integrins such as {alpha}1ß1 bind collagen and laminin, {alpha}5ß1 binds fibronectin, and {alpha}vß1 binds vitronectin and fibronectin. However, {alpha}1, {alpha}5, and {alpha}v mRNA could not be studied due to the lack of specific rat probes and the inability of the human probes to hybridize with rat mRNA.

To be able to assay for more integrin subunits in bone and examine integrin protein levels, Western blot analysis was performed on bones cultured for 96 h with varying concentrations of ascorbic acid (Fig. 5Go). The relative density of each integrin subunit was plotted and compared with control bones treated with 100 µg/ml ascorbic acid, which was arbitrarily set at 100. The Western blots demonstrated a significant, dose-dependent decrease in {alpha}1, {alpha}2, and ß1 protein in parietal bones cultured with varying concentrations of ascorbate. The {alpha}3 and {alpha}5 integrin subunits were not significantly affected by ascorbic acid. The {alpha}v and ß3 subunits were slightly decreased in bones cultured without ascorbic acid, whereas no ß5 was detected in fetal rat parietal bones.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 5. Western blots of integrin subunits from bones treated with 0, 1, 10, and 100 µg/ml ascorbic acid for 96 h (A) and their relative densities (B). Three experiments.

 
Immunofluorescence staining of the bone confirmed the concentration-dependent decrease in {alpha}2 and ß1 integrin subunits at 96 h of culture (Fig. 6Go). In control bones, staining for both subunits was found primarily in the osteoblast layer overlying the bone (Figs. 6AGo and C). Weak ß1 staining was also seen in the cells of the periosteum (Fig. 6AGo). Immunofluorescent labeling of ß1 and {alpha}2 was markedly diminished in a dose-dependent manner in bones treated with 0, 1, and 10 µg/ml ascorbic acid in comparison with the control bones with 100 µg/ml ascorbic acid. Only bones treated with 1 and 100 µg/ml ascorbic acid are illustrated in Fig. 6Go. The control bones incubated with nonimmune serum showed little or no staining (Fig. 6EGo).



View larger version (62K):
[in this window]
[in a new window]
  		Figure 6. Immunofluorescence light micrographs of fixed, frozen parietal bones treated for 96 h with 100 (A and C)) and 1 (B and D) µg/ml ascorbic acid. A, ß1 integrin was found mainly in the osteoblast layer (arrow) above mineralized matrix (M) and to a lesser extent in periosteum (p). B, There was a marked decrease in ß1 staining in the osteoblast layer and periosteum. C, {alpha}2 staining was found mainly in the osteoblast layer (arrow). D, Ascorbic acid (1 µg/ml) decreased {alpha}2 staining. E, A control bone treated with nonimmune serum had little or no staining. Magnification, x210; bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In fetal rat parietal bones, ascorbic acid deficiency inhibited calcification, but did not affect type I collagen message or protein levels at 96 h. However, collagen was significantly underhydroxylated in bones treated with 0, 1, and 10 µg/ml ascorbic acid compared with control bones with 100 µg/ml. Light microscopic examination of the bones deficient in ascorbic acid demonstrated an irregular-shaped mineralizing front with a disrupted layer of misshapen osteoblasts. Integrins, which are involved in cell signaling and attachment to the extracellular matrix, were down-regulated in a dose-dependent manner in ascorbic acid-deficient bones. Specifically, the protein levels of the integrin subunits that form the collagen receptors, {alpha}1ß1 and {alpha}2ß1, were decreased, whereas {alpha}3, {alpha}5, and {alpha}v were not significantly affected. Message levels for the integrin subunits {alpha}2 and ß1 were also inhibited with no change in {alpha}3 mRNA levels.

Changes in message and protein levels of the collagen integrins preceded any changes in collagen synthesis. These data suggest that integrin synthesis is regulated independently of the synthesis of their attachment proteins. Instead, integrin synthesis may be modulated by engagement of the integrin with its substrate. In bone culture, the integrins involved in binding collagen and not other matrix proteins, were down-regulated, which demonstrates the specificity of integrin function and regulation. If integrins are able to respond rapidly and specifically to a change in the organization of the matrix, then they may be a useful therapeutic tool to detect alterations in matrix structure in metabolic bone diseases, such as osteogenesis imperfecta. Mutations in pro{alpha}1 and pro{alpha}2 chains of type I collagen have been shown to cause osteogenesis imperfecta (40, 41), however, the integrins for collagen have not been studied in these patients.

The decrease in calcification associated with ascorbic acid deficiency in our system may be attributed to the improper formation of collagen fibrils, which may directly decrease the ability of the matrix to calcify, and/or may be due to an effect on integrin expression, which affects osteoblast function, leading to the decrease in mineralization. Ascorbic acid is necessary for the hydroxylation of collagen, and the presence of hydroxyproline is essential to stabilize the triple helix of two {alpha}1 and one {alpha}2 chains to form collagen fibrils. Because type I collagen accounts for approximately 90% of the organic matrix of bone, defects in collagen synthesis or assembly profoundly affect the structure of bone. There are a large number of matrix proteins that bind collagen such as decorin, osteonectin, fibronectin, thrombospondin, type V collagen, and vitronectin, among others, and therefore, the improper assembly of collagen fibrils may lead to the inability of some of these proteins to bind collagen and to nucleate hydroxyapatite deposition (42). Interestingly, the {alpha}vß3 that binds the bone matrix protein osteopontin was the only other integrin, besides the collagen integrins, that was decreased. (The {alpha}vß3 also binds fibrinogen, von Willebrand factor, and vitronectin.) However, {alpha}vß3 was decreased only in bones cultured without ascorbic acid. Osteopontin has been shown by immunogold electron microscopy to be extensively associated with collagen fibrils (43). Therefore, perhaps the underhydroxylated collagen fibrils are also defective in their ability to interact with other extracellular matrix proteins such as osteopontin, which leads to a decrease in calcification. The underhydroxylated collagen molecules appeared to have been maintained in the matrix, because there was no increase in [3H]proline labeling in the supernatant of the bone cultures nor a decrease in [3H]proline labeling of the cell layer.

Osteoblast activity may also be impaired by ascorbic acid deficiency due to the accumulation of nonhelical, underhydroxylated procollagen in their rough endoplasmic reticulum, as has been found in chondrocytes (44). The accumulation of underhydroxylated procollagen within cells may affect the osteoblast’s ability to secrete bone matrix proteins and to synthesize and process other proteins essential for osteoblast function. However, collagen continued to be synthesized at the same rate in bones with decreased ascorbic acid levels compared with bones with 100 µg/ml ascorbic acid.

Finally, the defective collagen fibrils may not be recognized by the integrins on the surface of the osteoblast due to conformational changes in collagen. Precedence for this hypothesis is found in studies in which the triple helical structure of collagen VI appeared to be important for adhesion of various cell lines (29). The amount of {alpha}2 integrins on MG-63 and HOS cells also affected the ability of the cells to contract collagen gels (45), whereas {alpha}1ß1 has been shown to mediate collagen matrix reorganization by myofibroblasts after injury (46). Thus, integrins on osteoblasts may be unable to bind to the underhydroxylated collagen and are down-regulated, leading to a loss of cell adhesion and osteoblast organization. Because osteoblast secretion of bone matrix proteins is polarized, the loss of osteoblast organization may lead to disorganized matrix assembly and decreased bone formation. Unfortunately, there is no information on how the disruption of physical integrity of collagen fibrils by the formation of underhydroxylated type I collagen may modify integrin binding and expression. Integrin function appears to be essential for bone formation, as shown by experiments in which synthetic peptides containing a RGD sequence, competitive inhibitors of integrins, were able to inhibit mineralization in fetal rat bone cultures (47). The control, inactive RAD peptide, had no effect on calcification. Interestingly, osteoblast organization was disrupted and collagen synthesis was not affected, similar to our findings with decreased concentrations of ascorbic acid.

The effect of ascorbic acid on extracellular matrix proteins and their receptors along with an effect on alkaline phosphatase levels (8, 9) and cell proliferation (6, 7) in bone cell cultures, has led to the hypothesis that there is a matrix-generated signal that is dependent on collagen organization and the presence of ascorbic acid (8, 9). Without these signals, or with inappropriate signals, the subsequent induction of osteoblast differentiation and calcification may be impaired, leading to a disruption of the osteoblast layer and a decrease in mineralization, as was found in this bone organ culture system and in swine in vivo with ascorbic acid deficiency (48). In swine, ossification was not only deranged but also there were fewer osteoblasts and a loosening of the periosteum from the cortex, which suggests defective integrin function, because integrins maintain tissue integrity. In addition to ascorbic acid’s effect on osteoblasts, it may also affect other cells in bone. Immunofluorescence microscopy of {alpha}2ß1 was not only seen in the osteoblast layer but also in the periosteum. Thus, a decrease in {alpha}2ß1 in other cells may affect their function. With prolonged ascorbic acid deficiency, collagen protein levels are inhibited, which would further impair bone formation (49, 50). Thus, ascorbic acid is essential for normal bone formation due to its effects on collagen hydroxylation and integrins for collagen.


   Footnotes
 
1 This work was supported by a high school research fellowship from the American Heart Association Connecticut Affiliate, Inc. (to D.F.G.) and a NIH Grant AR42367 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (to G.G.). Back

Received March 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hausmann E 1967 Cofactor requirements for the enzymatic hydroxylation of lysine in a polypeptide precursor of collagen. Biochim Acta 133:591–593
  2. Kivirikko KI, Prockop DJ 1972 Partial purification and characterization of protocollagen lysine hydroxylase from chick embryos. Biochim Biophys Acta 258:366–379[Medline]
  3. Dixon S, Kulaga A, Jaworski E, Wilson J 1991 Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors. J Bone Miner Res 6:623–629[Medline]
  4. Franceschi RT, Wilson JX, Dixon SJ 1995 Requirement for Na+ dependent ascorbic acid transport in osteoblast function. Am J Physiol 268:C1430–C1439
  5. Reynolds J 1967 The synthesis of collagen by chick bone rudiments in vitro. Exp Cell Res 47:42–48[CrossRef][Medline]
  6. Harada S, Matsumoto T, Ogata E 1991 Role of ascorbic acid in the regulation of proliferation in osteoblast-like MC3T3–E1 cells. J Bone Miner Res 9:903–908
  7. Spindler K, Shapiro D, Gross S, Brighton C, Clark C 1989 The effect of ascorbic acid on the metabolism of rat calvarial bone cells in vitro. J Orthop Res 7:696–701[CrossRef][Medline]
  8. Franceschi R, Iyer B 1992 Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3–E1 cells. J Bone Miner Res 7:235–246[Medline]
  9. Franceschi R, Iyer B, Cui Y 1994 Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3–E1 cells. J Bone Miner Res 9:843–854[Medline]
  10. Asahira I, Sampath TK, Nishimura I, Hauschka PV 1993 Human osteogenic protein-1 induces both chondroblastic cells derived from newborn rat calvaria. J Cell Biol 93:921–933[Abstract/Free Full Text]
  11. Choong P, Martin T, Ng K 1993 Effects of ascorbic acid, calcitriol and retinoic acid on the differentiation of preosteoblasts. J Orthop Res 11:638–647[CrossRef][Medline]
  12. Franceschi RT, Young J 1990 Regulation of alkaline phosphatase by 1,25-dihydroxy-vitamin D3 & ascorbic acid in bone-defined cells. J Bone Miner Res 5:1157–1167[Medline]
  13. Ishida H, Bellows C, Aubin J, Heersche J 1993 Characterization of the 1,25 (OH)2 D3-induced inhibition of bone nodule formation in long-term cultures of fetal rat calvaria cells. Endocrinology 132:61–66[Abstract]
  14. Clark E, Brugge J 1995 Integrins and signal transduction pathways: the road taken. Science 268:233–239[Abstract/Free Full Text]
  15. Hynes R 1992 Integrins: versatility, modulation and signaling in cell adhesion. Cell 69:11–25[CrossRef][Medline]
  16. Ruoslahti E 1991 Integrins. J Clin Invest 87:1–5
  17. Piersbacher M, Ruoslahti E 1984 Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309:497–500
  18. Yamada K, Kennedy D 1984 Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J Cell Biol 99:29–36[Abstract/Free Full Text]
  19. Puleo DA, Bizios R 1991 RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion. Bone 1991:271–276
  20. Burridge K, Faith K, Kelly Y, Nuckolls G, Turner C 1988 Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4:487–525[CrossRef]
  21. Horwitz A, Duggan K, Buck C, Beckerle M, Burridge K 1986 Interaction of plasma membrane receptor with talin: a transmembrane linkage. Nature 320:531–533[CrossRef][Medline]
  22. Singer I I 1982 Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts. J Cell Biol 92:398–408[Abstract/Free Full Text]
  23. Richardson A, Parson J 1995 Signal transduction through integrins: a central role for focal adhesion kinase? Bioessays 17:229–236[CrossRef][Medline]
  24. Schaller M, Hildebrand J, Shannon J, Fox J, Vines R, Parsons J 1994 Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 14:1680–1688[Abstract/Free Full Text]
  25. Grzesik W, Robey P 1994 Bone matrix RGD glycoproteins: immunolocalization and interaction with primary human primary osteoblastic cells in vitro. J Bone Miner Res 9:4487–496
  26. Hughes D, Salter D, Dedhar S 1993 Integrin expression in human bone. J Bone Miner Res 8:527–533[Medline]
  27. Saito T, Albelda S, Brighton C 1994 Identification of integrin receptors on cultured human bone cells. J Orthop Res 12:384–394[CrossRef][Medline]
  28. Clover J, Dodds R, Gowen M 1992 Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture. J Cell Sci 103:267–271[Abstract/Free Full Text]
  29. Pfaff M, Aumalley M, Specks U, Knolle J, Zerwes H, Timpl R 1993 Integrin and Arg-Gly-Asp dependence of cell adhesion to the native and unfolded triple helix of collagen Type VI. Exp Cell Res 206:167–176[CrossRef][Medline]
  30. Gronowicz G, McCarthy MB 1995 Glucocorticoids inhibit osteoblast adhesion to extracellular matrix proteins and ß1 integrin expression. Endocrinology 136:598–608[Abstract]
  31. Gronowicz G, DeRome M, McCarthy M 1991 Glucocorticoids inhibit fibronectin synthesis and mRNA levels in cultured fetal rat parietal bones. Endocrinology 128:1107–114[Abstract]
  32. Gohel A, Hand A, Gronowicz G 1995 Immunogold localization of B1-integrin in bone: effect of glucocorticoids and insulin-like growth factor I on integrins and osteocyte formation. J Histochem Cytochem 43:1085–1096[Abstract]
  33. Gronowicz G, Woodiel FN, McCarthy M-B, Raisz LG 1989 In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of ß-glycerol phosphate. J Bone Miner Res 4:313–324[Medline]
  34. Kissane J, Robins E 1958 The fluorimetric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J Biol Chem 233:184–188[Free Full Text]
  35. Peterkofsky B, Diegelmann R 1971 Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. Biochem 10:988–994[CrossRef][Medline]
  36. Peterkofsky B, Diegelmann R 1972 Collagen biosynthesis during connective tissue development in chick embryo. Dev Biol 28:443–453[Medline]
  37. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  38. Genovese C, Rowe D, Kream B 1984 Construction of DNA sequences complementary to rat {alpha}1 and {alpha}2 collagen mRNA and their use in studying the regulation of Type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23:6210–6216[CrossRef][Medline]
  39. Thomas P 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201–5205[Abstract/Free Full Text]
  40. Prockop DJ, Colige A, Helminen H, Khillan JS, Pereira R, Vandenberg P 1993 Mutations in type I procollagen that cause osteogenesis imperfecta: effect of the mutations on the assembly of collagen into fibrils, the basis of phenotypic variations, and potential antisense therapies. J Bone Miner Res 8:489–492
  41. Shapiro JR, Primorac D, Rowe D W 1996 Osteogenesis imperfecta: current concepts. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, New York, pp 889–902
  42. Robey G H 1996 Bone matrix proteoglycans and glycoproteins. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, New York, pp 155–165
  43. McKee M, Nanci A, Landis W, Gotoh Y, Gerstenfeld L, Glimcher M 1991 Developmental appearance and ultrastructural immunolocalization of a major 66 kDa phosphoprotein in embryonic and post-natal chicken bone. Anat Rec 228:77–92
  44. Pacifici M, Iozzo R 1988 Remodeling of the rough endoplasmic reticulum during stimulation of procollagen secretion by ascorbic acid in cultured chondrocytes. J Biol Chem 263:2483–2492[Abstract/Free Full Text]
  45. Riikonen T, Koivisto L, Vihinen P, Heino J 1995 Transforming growth factor-B regulates collagen gel contraction by increasing {alpha}2ß1 integrin expression in osteogenic cells. J Biol Chem 270:376–382[Abstract/Free Full Text]
  46. Gotwals P, Chi-Rosso G, Lindner V, Yang J, Ling L, Fawell S, Koteliansky V 1996 The {alpha}1ß1 integrin is expressed during neointima formation in rat arteries and mediates collagen matrix reorganization. J Clin Invest 97:2469–2477[Medline]
  47. Gronowicz G, DeRome M 1994 A synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones. J Bone Miner Res 9:193–202[Medline]
  48. Wegger I, Palludan B 1994 Vitamin C deficiency causes hematological and skeletal abnormalities during fetal development in swine. Nutrient Metab 124:241–248
  49. Chen T, Raisz L 1975 The effects of ascorbic acid deficiency on calcium and collagen metabolism in cultured fetal rat bones. Calcif Tissue Res 17:113–128[Medline]
  50. Denis I, Pointillart A, Lieberherr M 1994 Cell stage-dependent effects of ascorbic acid on cultured porcine bone cells. Bone Miner 25:149–161[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ganta, D. R.
Right arrow Articles by Gronowicz, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ganta, D. R.
Right arrow Articles by Gronowicz, G. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals