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Ascorbic acid induction of chondrocyte maturation
242
Bone and Mineral, 17 (1992) 242-246
Elsevier This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles. held November 16-20,1991, Hilton Head, South Carolina.
Ascorbic acid inductionof chondrocyte maturation
P.S. Leboy’, T.A. Sullivan’, A.S. Menko’ancl
M.
‘Department of Biochemistry, School of Dental Medicine, and 2Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Kay wools: Chondrocytes; Alkaline phosphatase; Ascorbic acid; Lacticacid; Cell attachment
Introduction During endochondral bone formation, cells in the cartilage undergo an orderly series of changes including hypertrophy, a marked elevation in alkaline phosphatase activity, and a switch from synthesis of type II and IX collagens to type X collagen [l-3]. In chick embryos, chondrocytes from the cephalic portion of the sternum (upper sternal chondrocytes) undergo these changes at days 17-18; thus, upper sternal chondrocytes from 14 day embryos are ‘pre-hypertrophic’ [4,5]. These prehypertrophic chondrocytes, when cultured as monolayers in DMEM -t- 10% FCS (control medium), retain their chondrogenic phenotype for several weeks, synthesizing type II colagen and showing limited alkaline phosphatase activity or type X collagen synthesis. Exposure to ascorbic acid (Asc) induces a 15-20~ increase in alkaline phosphatase activity and a shift from type II to type X synthesis [6]. As a first step towards understanding the mechanism by which Asc induces these changes in gene expression, we have explored other changes associated with Asc treatment of pre-hypertrophic chondrocytes. We present here evidence and Asctreated cultures also show changes in metabolism and in cell-matrix interactions.
Methodsand Materials Cells from the cephalic portion of 14 day chick embryo sternae were cultured as described previously (61; in some experiments, 4 units/ml hyaluronidase was added to promote cell attachment. After 1 day, Asc (lOpg/ml) was added to some cultures; the Asc concentration was increased to 25 pg/ml3 days later. Alkaline phosphatase activity was assayed as described previously [6]. Cell-associated alkaline phosphatase was measured in extracts prepared by incubating washed cells layers in buffer
containing Triton-X100 at 37°C for 30 min. Total alkaline phosphatase activity was determined by resuspending the attached matrix in the Triton-containing cell extract. Lactate levels in culture medium were determined spectrophotometrically by deproteinizing an aliquot of medium with 2 volumes of 10% TCA and measuring NADH production at 340 nm in the presence of NAD and lactic dehydrogenase (Sigma). Collagen type I substrate was prepared by coating tissue culture dishes with Rthin film of Vitrogen (Collagen Biomedical). Tissue culture dishes were coated with collagens by adding a solution containing 0.6 mg collagen/ml to the dishes, incubating at room temperature for 2 min and then aspirating off excess solution. The dishes were air dried and washed 3 x with P
Like many embryonic cells in culture, sternal chondrocytes produce lactic acid which accumulates in the medium. As cell density increases, the quantity of lactate released over several days is sufficient to change the color of phenol red-containing medium from pink to yellow. owever, chondrocytes cultured in the presence of Asc did not show such a color nge. We therefore analyzed levels of lactate accumulated in medium of cultures with and without ascorbate. By day 7 of culture, medium from control cultures accumulated 16.0 + 3.2 mM lactate in a 24 h period, while medium from Asc-treated cultures contained only 1.5 f 1.0 m examine the time course for Asc-induced changes in metabolism, Asc (10 ,ug/ml) was added at day 1 of culture and medium lactate assayed at 6,24,48 and 72 h. As shown in Fig. 1, decreased lactate accumulation in the medium of Asc-treated cultures was evident at 27 h with more pronounced effects at 48 and 72 h. This time course was similar to that seen for Asc-induced effects on alkaline phosphatase and type X mRNA induction [6]. These observations are consistent with earlier results of Ramp and Thornton [7],
0
20 40 60 hours after ascorbot
a0
Fig. I. Rate of appearanceof lactic acid in the medium of chondrocyte cultures in the absence of ascot-
bate (9-O)
or after addition of 10,ugImlascorbicacid at time 0 (
244 showing increased 0, consumption and decreased lactic acid production when Asc was added to cultured chick tibia. In vivo, a decreased lactic acid build-up would lead to increased pH of the extracellular matrix, thereby promoting mineral deposition. However, direct measurement of redox status in chick growth plate indicates decrease oxidative metabolism in the hypertrophic zone [8], suggesting that chondrocyte maturation in the growth plate may be associated with more rather than less glycolysis and lactic acid production. It is clear that further studies are needed to elucidate the effects of Asc on metabolism of cultured chondrocytes and their relationship to events occurring in viva. Cell adherence When cultured in bacterial Petri dishes, pre-hypertrophic chondrocytes remain in suspension with or without Asc. The suspended cells, like monolayer cultures, show rapid chsndrocyte maturation in the presence of Asc but not in control medium. Chondrocytes plated in tissue culture dishes with control medium will attach after either treatment with hyaluronidase or addition of Asc; in the absence of hyaluronidase or Asc most cells remain suspended. Cells attached with hyaluronidase form monolayers of polygonal cells while Asc-treated cells have a more rounded morphology. These results suggest either that the more collagen-rich matrix synthesized in the presence of Asc alters cell-matrix interaction or that Asc alters the properties of cell surface receptors. Chondrocytes plated on type I collagen showed increased cell attachment and spreading; almost 100% of the cells attached even without hyaluronidase treatment. However, like cells plated on plastic, chondrocytes on type I collagen still require Asc for induction of elevated alkaline phosphatase and type X collagen, and show a more rounded morphology in the presence of Asc. Thus, Asc effects on chondrocyte maturation cannot be duplicated merely by providing a collagen substrate. The mechanism of chondrocyte attachment to collagen substrate in the presence
CSAT:
-
+
-
+
Fii. 2. Attachmentof chondrocytesto type I collagen substrate in the absence and presence of ascorbate and antibody to& integrin.-Cells were cultured with and without ascorbate or CSATantibody;after48 h the numbers of attached vs. non-adherent cells were counted.
245
Localization of alkaline phosphatase activity in cultured chondrocytes. Cell-associatedalkaline phosphataseactivity (% of total activity) Origin of chondrocytes
Control
+ Ascorbate
+ Ascorbate + &glyceroPO,
Cephalic sternum Tibia1epiphysis
70 f 14 [n = 91 66rt: 14[n=7]
65 f 13 [n = 91 63 f 13 [n = 61
7090f12(n=4] 765~ S [n = 4)
and absence of Asc was examined by adding a monoclonal antibody raised against fil integrin, CSAT [9]. In the absence of Asc, this antibody blocked cell attachment to collagen (Fig. 2), demonstrating that attachment in control medium is mediated by &-containing integrins. Nowever, when Asc was added simultaneously with CSAT antibody a high proportion of the cells attached to the collagen substrate (Fig. 2), and when Asc was added prior to CSAT addition CSAT did not release the attached cells. These results suggest that Asc-induced cell attachment is not via #?, integrins. The transition from CSAT-sensitive attachment in control cultures to CSAT-insensitive attachment in ascorbate-treated cultures might be due to appearance of a new substrate attachment receptor in Asc-treated chondrocytes, or to Asc induction of matrix proteins which interact with chondrocytes via non-& integrin receptors. Alternatively, Asc may cause changes in B1integrins which impair CSAT recognition. We are currently exploring these hypotheses. Since Asc was observed to alter both chondrocyte morphology and attachment to matrices, we examined the possibility that Asc would alter release of alkaline phosphatase from cells. Cell-associated alkaline phosphatase was assayed by measuring enzyme activity in extracts prepared by treating the cell layer with buffer containing 1% Triton X-100. Total alkaline phosphatase activity in the cell layer was measured by seraping cells + matrix into buffer containing Triton. Membrane vesicle-associated alk,Jine phosphatase released into the medium was determined in pellets obtained by laigh speed cf-strifugation of the medium; ~5% of the activity was found in this fraction. Approximately two-thirds of the enzyme activity was cell-associated, wh&her Asc was present or absent (Table I). Thus, Asc effects are not associated wiih altered alkaline phosphatase localization.
This work was supported by NIEI Grant AR40075 to P.S.L. and Grants CA16502 and CA49866 to Dr. David Boettinger.
246 References 1 Castagnola P, Dozin B, Moro G, Cancedda R. Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro. J Cell Biol 1988;106:461-467. 2 Oshima 0, Leboy PS, McDonald SA, Tuan RS, Shapiro IM. Developmental expression of genes in chick growth cartilage detected by in situ hydridization. Calc Tiss Int 1989;45:182-192. 3 Leboy PS, Shapiro IM, Uschmann BD, Oshima 0, Lin D. Gene expression in mineralizing chick epiphyseal cartilage. J Biol Chem 1988;263:8SlS- 1520. 4 Hayashi M, Ninomiya Y, Parsons J, Hayashi K, Trelstad R, Olsen BR. Localization of mRNAs for collagen types I, II, IX and X in embryonic chick sterna by in situ hybridization. J Cell Biol 1985;101:94a(abst). S Reginato AM, Lash JW, Jimenez SA. Biosynthetic expression of type X collagen in embryonic chick sternum cartilage during development. J Biol Chem 1986;261:2897-2904. 6 Leboy PS, Vaias L, Uschmann B, Golub E, Adams SL, Pacifici M. Ascorbic acid induces alkaline phosphatase, type X collagen and calcium deposition in cultured chick chondrocytes. J Biol Chem 1989;264:17281-17286. 7 Ramp WK, Thornton PA. The effect of ascorbic acid on the glycolytic and respiratory metabolism of embryonic chick tibias. Calc ‘PissRes 1968;2:77-82. 8 Kakuta S, Golub E, Haselgrove JC, Chance B, Frasca P, Shapiro IM. Redox studies of the epiphy seal growth cartilage: pyridine nucleotide metabolism and the development of mineralization. J Bone Min Res 1986;1:433-440. 9 Horwitx A, Duggan K, Greggs R, Decker C, Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J Cell Biol198S;101:2134-2144.
Bone and Mineral, 17 (1992) 242-246
Elsevier This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles. held November 16-20,1991, Hilton Head, South Carolina.
Ascorbic acid inductionof chondrocyte maturation
P.S. Leboy’, T.A. Sullivan’, A.S. Menko’ancl
M.
‘Department of Biochemistry, School of Dental Medicine, and 2Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Kay wools: Chondrocytes; Alkaline phosphatase; Ascorbic acid; Lacticacid; Cell attachment
Introduction During endochondral bone formation, cells in the cartilage undergo an orderly series of changes including hypertrophy, a marked elevation in alkaline phosphatase activity, and a switch from synthesis of type II and IX collagens to type X collagen [l-3]. In chick embryos, chondrocytes from the cephalic portion of the sternum (upper sternal chondrocytes) undergo these changes at days 17-18; thus, upper sternal chondrocytes from 14 day embryos are ‘pre-hypertrophic’ [4,5]. These prehypertrophic chondrocytes, when cultured as monolayers in DMEM -t- 10% FCS (control medium), retain their chondrogenic phenotype for several weeks, synthesizing type II colagen and showing limited alkaline phosphatase activity or type X collagen synthesis. Exposure to ascorbic acid (Asc) induces a 15-20~ increase in alkaline phosphatase activity and a shift from type II to type X synthesis [6]. As a first step towards understanding the mechanism by which Asc induces these changes in gene expression, we have explored other changes associated with Asc treatment of pre-hypertrophic chondrocytes. We present here evidence and Asctreated cultures also show changes in metabolism and in cell-matrix interactions.
Methodsand Materials Cells from the cephalic portion of 14 day chick embryo sternae were cultured as described previously (61; in some experiments, 4 units/ml hyaluronidase was added to promote cell attachment. After 1 day, Asc (lOpg/ml) was added to some cultures; the Asc concentration was increased to 25 pg/ml3 days later. Alkaline phosphatase activity was assayed as described previously [6]. Cell-associated alkaline phosphatase was measured in extracts prepared by incubating washed cells layers in buffer
containing Triton-X100 at 37°C for 30 min. Total alkaline phosphatase activity was determined by resuspending the attached matrix in the Triton-containing cell extract. Lactate levels in culture medium were determined spectrophotometrically by deproteinizing an aliquot of medium with 2 volumes of 10% TCA and measuring NADH production at 340 nm in the presence of NAD and lactic dehydrogenase (Sigma). Collagen type I substrate was prepared by coating tissue culture dishes with Rthin film of Vitrogen (Collagen Biomedical). Tissue culture dishes were coated with collagens by adding a solution containing 0.6 mg collagen/ml to the dishes, incubating at room temperature for 2 min and then aspirating off excess solution. The dishes were air dried and washed 3 x with P
Like many embryonic cells in culture, sternal chondrocytes produce lactic acid which accumulates in the medium. As cell density increases, the quantity of lactate released over several days is sufficient to change the color of phenol red-containing medium from pink to yellow. owever, chondrocytes cultured in the presence of Asc did not show such a color nge. We therefore analyzed levels of lactate accumulated in medium of cultures with and without ascorbate. By day 7 of culture, medium from control cultures accumulated 16.0 + 3.2 mM lactate in a 24 h period, while medium from Asc-treated cultures contained only 1.5 f 1.0 m examine the time course for Asc-induced changes in metabolism, Asc (10 ,ug/ml) was added at day 1 of culture and medium lactate assayed at 6,24,48 and 72 h. As shown in Fig. 1, decreased lactate accumulation in the medium of Asc-treated cultures was evident at 27 h with more pronounced effects at 48 and 72 h. This time course was similar to that seen for Asc-induced effects on alkaline phosphatase and type X mRNA induction [6]. These observations are consistent with earlier results of Ramp and Thornton [7],
0
20 40 60 hours after ascorbot
a0
Fig. I. Rate of appearanceof lactic acid in the medium of chondrocyte cultures in the absence of ascot-
bate (9-O)
or after addition of 10,ugImlascorbicacid at time 0 (
244 showing increased 0, consumption and decreased lactic acid production when Asc was added to cultured chick tibia. In vivo, a decreased lactic acid build-up would lead to increased pH of the extracellular matrix, thereby promoting mineral deposition. However, direct measurement of redox status in chick growth plate indicates decrease oxidative metabolism in the hypertrophic zone [8], suggesting that chondrocyte maturation in the growth plate may be associated with more rather than less glycolysis and lactic acid production. It is clear that further studies are needed to elucidate the effects of Asc on metabolism of cultured chondrocytes and their relationship to events occurring in viva. Cell adherence When cultured in bacterial Petri dishes, pre-hypertrophic chondrocytes remain in suspension with or without Asc. The suspended cells, like monolayer cultures, show rapid chsndrocyte maturation in the presence of Asc but not in control medium. Chondrocytes plated in tissue culture dishes with control medium will attach after either treatment with hyaluronidase or addition of Asc; in the absence of hyaluronidase or Asc most cells remain suspended. Cells attached with hyaluronidase form monolayers of polygonal cells while Asc-treated cells have a more rounded morphology. These results suggest either that the more collagen-rich matrix synthesized in the presence of Asc alters cell-matrix interaction or that Asc alters the properties of cell surface receptors. Chondrocytes plated on type I collagen showed increased cell attachment and spreading; almost 100% of the cells attached even without hyaluronidase treatment. However, like cells plated on plastic, chondrocytes on type I collagen still require Asc for induction of elevated alkaline phosphatase and type X collagen, and show a more rounded morphology in the presence of Asc. Thus, Asc effects on chondrocyte maturation cannot be duplicated merely by providing a collagen substrate. The mechanism of chondrocyte attachment to collagen substrate in the presence
CSAT:
-
+
-
+
Fii. 2. Attachmentof chondrocytesto type I collagen substrate in the absence and presence of ascorbate and antibody to& integrin.-Cells were cultured with and without ascorbate or CSATantibody;after48 h the numbers of attached vs. non-adherent cells were counted.
245
Localization of alkaline phosphatase activity in cultured chondrocytes. Cell-associatedalkaline phosphataseactivity (% of total activity) Origin of chondrocytes
Control
+ Ascorbate
+ Ascorbate + &glyceroPO,
Cephalic sternum Tibia1epiphysis
70 f 14 [n = 91 66rt: 14[n=7]
65 f 13 [n = 91 63 f 13 [n = 61
7090f12(n=4] 765~ S [n = 4)
and absence of Asc was examined by adding a monoclonal antibody raised against fil integrin, CSAT [9]. In the absence of Asc, this antibody blocked cell attachment to collagen (Fig. 2), demonstrating that attachment in control medium is mediated by &-containing integrins. Nowever, when Asc was added simultaneously with CSAT antibody a high proportion of the cells attached to the collagen substrate (Fig. 2), and when Asc was added prior to CSAT addition CSAT did not release the attached cells. These results suggest that Asc-induced cell attachment is not via #?, integrins. The transition from CSAT-sensitive attachment in control cultures to CSAT-insensitive attachment in ascorbate-treated cultures might be due to appearance of a new substrate attachment receptor in Asc-treated chondrocytes, or to Asc induction of matrix proteins which interact with chondrocytes via non-& integrin receptors. Alternatively, Asc may cause changes in B1integrins which impair CSAT recognition. We are currently exploring these hypotheses. Since Asc was observed to alter both chondrocyte morphology and attachment to matrices, we examined the possibility that Asc would alter release of alkaline phosphatase from cells. Cell-associated alkaline phosphatase was assayed by measuring enzyme activity in extracts prepared by treating the cell layer with buffer containing 1% Triton X-100. Total alkaline phosphatase activity in the cell layer was measured by seraping cells + matrix into buffer containing Triton. Membrane vesicle-associated alk,Jine phosphatase released into the medium was determined in pellets obtained by laigh speed cf-strifugation of the medium; ~5% of the activity was found in this fraction. Approximately two-thirds of the enzyme activity was cell-associated, wh&her Asc was present or absent (Table I). Thus, Asc effects are not associated wiih altered alkaline phosphatase localization.
This work was supported by NIEI Grant AR40075 to P.S.L. and Grants CA16502 and CA49866 to Dr. David Boettinger.
246 References 1 Castagnola P, Dozin B, Moro G, Cancedda R. Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro. J Cell Biol 1988;106:461-467. 2 Oshima 0, Leboy PS, McDonald SA, Tuan RS, Shapiro IM. Developmental expression of genes in chick growth cartilage detected by in situ hydridization. Calc Tiss Int 1989;45:182-192. 3 Leboy PS, Shapiro IM, Uschmann BD, Oshima 0, Lin D. Gene expression in mineralizing chick epiphyseal cartilage. J Biol Chem 1988;263:8SlS- 1520. 4 Hayashi M, Ninomiya Y, Parsons J, Hayashi K, Trelstad R, Olsen BR. Localization of mRNAs for collagen types I, II, IX and X in embryonic chick sterna by in situ hybridization. J Cell Biol 1985;101:94a(abst). S Reginato AM, Lash JW, Jimenez SA. Biosynthetic expression of type X collagen in embryonic chick sternum cartilage during development. J Biol Chem 1986;261:2897-2904. 6 Leboy PS, Vaias L, Uschmann B, Golub E, Adams SL, Pacifici M. Ascorbic acid induces alkaline phosphatase, type X collagen and calcium deposition in cultured chick chondrocytes. J Biol Chem 1989;264:17281-17286. 7 Ramp WK, Thornton PA. The effect of ascorbic acid on the glycolytic and respiratory metabolism of embryonic chick tibias. Calc ‘PissRes 1968;2:77-82. 8 Kakuta S, Golub E, Haselgrove JC, Chance B, Frasca P, Shapiro IM. Redox studies of the epiphy seal growth cartilage: pyridine nucleotide metabolism and the development of mineralization. J Bone Min Res 1986;1:433-440. 9 Horwitx A, Duggan K, Greggs R, Decker C, Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J Cell Biol198S;101:2134-2144.