Effects of ascorbic acid, calcitriol, and retinoic acid on the differentiation of preosteoblasts

Journal of Orthopaedic Research 1k63S-647 The Journal of Bone and Joint Surgery, Inc. 0 1993 Orthopaedic Research Society

Effects of Ascorbic Acid, Calcitriol, and Retinoic Acid on the Differentiation of Preosteoblasts

Peter F. M. Choong, *T. John Martin, and *Kong Wah Ng

Departments of Orthopaedics and *Medicine, St. Vincent’s Institute of Medical Research, St. Vincent’s Hospital, Melbourne, Australia

Summary: The responses of the immortalized rat preosteoblast UMR-201-10B to ascorbic acid (AA), 1,25(OH),D3 (calcitriol), and retinoic acid (RA) were examined. UMR-201-1OB cells have an undetectable basal alkaline phosphatase (ALP) activity that is induced after 24 h of treatment with M R A (4.64 ? 0.06 pmol/h/mg of protein). The addition of M calcitriol resulted in a slight induction of ALP activity after 72 h (0.43 t 0.07 ymollhlmg of protein). When calcitriol was added to RA, however, over the same period ALP activity was enhanced significantly compared with treatment with RA alone (RA and calcitriol, 12.29 ? 0.86 pmol/h/mg of protein). Treatment with AA (50 pg/ml) alone had no effect on ALP activity but increased RA-induced ALP activity to 6.78 -C 0.28 pmol/h/mg of protein at 24 h. In contrast, AA inhibited calcitriol-induced ALP activity after 7 days of combined treatment with calcitriol (calcitriol, 7.73 -C 0.16 prnolihimg of protein; AA and calcitriol, 1.44 i 0.06 pmol/h/mg of protein). Individually, R A and calcitriol induced mRNA expression for ALP, matrix-gla protein (MGP), and osteopontin (OP). The steady state level of pro-al(1) collagen mRNA also was increased significantly by treatment with RA and AA individually. The combination of RA and calcitriol had a synergistic effect on ALP, OP, and especially MGP mRNA expression but significantly reduced the expression of p r o d (I) collagen mRNA. AA enhanced the effect of RA on the expression of pro-al(1) collagen, MGP, and ALP mRNAs as well as the effect of calcitriol on OP and MGP. The addition of AA to RA resulted in a decrease in the steady state level of OP, whereas its cotreatment with calcitriol caused a decrease in pro-al(1) collagen and ALP mRNA. In conclusion, these studies identify RA, calcitriol, and AA as regulators of differentiated osteoblast function.

The control of osteoblast development, differentiation, and function is multifactorial. Contributions from systemically active and locally generated factors, acting in an autocrine or paracrine manner, or

both, as well as through cell-cell and cell-matrix interactions, serve to highlight the complexity of osteoblast regulation.

1,25-Dihydroxyvitamin D3 (26,27,32) is one of sev- era1 systemically active factors that are known to modulate osteoblast differentiation. Osteosarcoma

Received February 7,1992; accepted January 27,1993. Please address correspondence and reurint reauests to Dr. lines treated with calcitriol have demonstrated

P. F. M. Choong at St. Vincent’s Institute if Medical Research, St. Vincent’s Hospital, 41 Victoria Parade, Melbourne, Victoria 3065, Australia.

Read in part at the first international Osteosarcoma Re- search Conference, Pittsburgh, PA, U.S.A., October 16-18,1991.

changes in growth and proliferation that are asso- ciated with an alteration in synthesis and function Of proteins such as osteocalcin (BGP) (25), alkaline phosphatase (ALP) (13,26,27), fibronectin (lOJl),

638

REGULATION OF PREOSTEOBLAST DIFFERENTIATION 639

matrix-Gla protein (MGP) (14), osteopontin (OP) (31,33), and type-I collagen (5,21). These studies have shown that calcitriol increases the steady state level of mRNA of these genes. In addition, others have postulated that calcitriol may regulate gene expression indirectly through a modulatory effect on extracellular matrix synthesis (10-12).

Ascorbic acid (AA) is a nutritionally derived factor that is a prerequisite for the normal hydroxyla- tion, processing, and secretion of collagen. AA has been shown to alter the in vitro differentiation of various cell lines including osteoblasts (20,37), fibroblasts (22), and chondrocytes (3,9,16). Like calcitriol, AA can modulate phenotypic expression of osteoblasts, as shown by the enhancement of ALP activity in certain rat calvaria-derived clonal cell lines (37). Although obviously important for normal bone metabolism, as evidenced by the physiological and clinical derangement seen in scorbutic animals and humans, the mechanism of its trophic action remains unclear. Several studies, however, have sought to link its central role in collagen metabolism and cellular adhesion to the effects on in vitro cellular differentiation and function (10,11,17,19,24), thus al- luding to the role of the extracellular matrix in reg- ulating osteoblast function.

In this study, we examined the effects of calcitriol and AA on retinoic acid (RA)-induced differentiated osteoblast function as well as the independent role of each as an inducer of osteoblast differentiation. These three agents have been studied, individually and in combination, for their effects on cell growth, ALP specific activity, and expression of genes for a number of major components of bone matrix.

A preosteoblastic calvaria-derived rat clonal cell line (29), UMR-201, was established by this labora- tory and later immortalized in two clonal cell lines (41), UMR-201-1OA and UMR-201-1OB, by stable transfection with simian virus 40 large T antigen. Previous studies (41) on this clonal line have demonstrated the constitutive expression of osteoblast phenotypic markers such as OP, type-I collagen, and osteonectin but not of ALP activity or mRNA. Stim- ulation with RA, however, enhances the expression of these genes and, in addition, induces the significant expression of ALP. This confirms the place of UMR-201-1OB in the osteoblast lineage and sup- ports its designation as a preosteoblast. The pleiotropic effects of calcitriol, AA, and RA on osteoblast differentiation were studied in the UMR-201-10B cells.

MATERIALS AND METHODS Materials

Alpha-modified Minimal Essential Medium (a- MEM) was purchased from Flow Laboratories Aus- tralasia (Mt. Waverley, Victoria, Australia). All-trans RA was purchased from Sigma Chemical (St. Louis, MO, U.S.A.). D-iso-ascorbic acid sodium salt was purchased from BDH Chemicals (Poole, England). [32P]a-CTP (specific activity 3,000 Ci/mmol) was purchased from Amersham Australia (Sydney, New South Wales, Australia). Molecular biology grade agarose was obtained from International Biotech- nologies (New Haven, CT, U.S.A.). Fetal bovine serum (FBS) is a product of Grand Island Biolog- ical Laboratories (Melbourne, Victoria, Australia). BCA protein assay reagents are a product of Pierce (Rockford, IL, U.S.A.), Kodak X-OMAT AR Film is a product of Eastman Kodak (Rochester, NY, U.S.A.), and UVL-21 Blackray lamp is a product of Ultraviolet Products (San Gabriel, CA, U.S.A.). All other reagents were of analytical grade and were obtained from standard suppliers.

Growth Studies

UMR-201-10B cells were subcultured into 9.6 cmz six-well plates (Flow Laboratories, McLean, VA, U.S.A.) in a-MEM with 10% FBS at a plating density of 5 x lo4 cells/well. Cultures were maintained at 37°C in a 5% C 0 2 environment. After 24 h, the medium was changed to a-MEM with 2% vitamin A-depleted FBS (A- FBS). To prepare A- FBS, serum was ex- posed to long-wave ultraviolet light at 366 Nm for 12 h to eliminate any endogenous RA (29). When the cells were approximately 80% confluent (3-4 days), the medium was changed and RA, calcitriol, and AA were added in varying combinations. The plates then were wrapped in aluminum foil to protect the RA from UV-induced degradation. Cells were harvested after 48 h with 1 ml of 0.0125% trypsin-0.5 mM sodium EDTA per well and were counted in a model DN Coulter Counter (Coulter Electronics, Luton, En- gland). Stock solutions of AA were made up fresh daily in sterile phosphate buffered saline (PBS) and subsequently diluted in culture medium to give the required final concentration of 50 pg/ml. Stock solutions of RA (10” M ) and calcitriol M ) were made up in absolute alcohol and stored at -70°C. The final concentrations of RA and calcitriol in medium were and lo-* M , respectively. This concentration

J Orthop Res, Vol. 11, No. S, 1993

640 P. F: M. CHOONG E T AL.

of RA previously has been shown to be maximally effective for inhibition of growth and the induction of ALP activity (41). Calcitriol at M also has been found to be the maximally effective concentration for growth inhibition of UMR-201-1OB cells (results not shown). Control cells received an equal amount of absolute alcohol so that the final concentration of alcohol in the medium was 0.001%.

ALP Activity

ALP activity was measured by the hydrolysis of p-nitrophenyl phosphate, as previously described (29). Briefly, ALP produced by cells in culture hydro- lyses exogenous p-nitrophenyl phosphate, and the subsequent release of p-nitrophenol is accompanied by a colour change that can be measured spectropho- tometrically at 410 Nm. The specific activity of ALP was expressed as ymoles of p-nitrophenol released per hour per mg of protein. Spectrophotometric quantitation of protein concentration was performed at 562 Nm, as previously described (36), with bovine serum albumin used as standards. Briefly, the addition of Cu2+ to protein in an alkaline medium yields Cu", which chelates to bicinchoninic acid (BCA). The purple protein-Cu"-BCA complex exhibits a strong absorbance at 562 Nm, thus allowing spectrophotometric quantitation of protein in aqueous solution. In the experiments, cells were subcultured into 9.6 cm2 six-well plates at a density of 5 x lo4 cells/well in a-MEM with 10% FBS and cultured at 37°C in a 5% C02 air environment. After 24 h, the medium was changed to a-MEM with 2% A- FBS. Cells were treated with RA M ) , calcitriol (lo-* M ) , and AA (50 ,ug/ml) alone and in combination, as described for individual experiments. The medium was changed every 48 h with fresh additions of RA and calcitriol, and AA was added daily.

Northern Blot Analysis

Cells were subcultured into 180 cm2 tissue culture flasks at a seeding density of 9.4 x lo5 cells/cm2 in a-MEM with 10% FBS at 37°C in a 5% C 0 2 air environment. The medium then was replaced with a-MEM with 2% A- FBS and RA M ) , calcitriol

M ) , and AA (50 yg/ml), added alone or in combination, and the cells were incubated for 7 days. The medium was changed every 48 h with fresh additions of RA and calcitriol, and AA was replaced daily.

Total RNA was isolated with the guanidine thio-

cyanate method (8); briefly, this consists of chemical cell lysis, followed by phenol-chloroform-isoamyl alcohol extraction and finally, propanol precipitation of RNA. Total RNA extracted then is separated in a 1.5% agarose/formaldehyde gel and transferred to nylon filters (Amersham, Poole, England). Filters were prehybridised for 1 h at 42°C in buffer containing 50% formamide, 5-fold SSPE (SSPE contains 0.15 M NaCl, 0.01 M NaH2P0,, and 0.001 MEDTA), 5-fold Denhardt's solution, 0.5% sodium dodecyl sulphate (SDS), and 1% skimmed milk. Next, filters were placed in fresh buffer and specific [32P]a-dCTP labeled complementary DNA probes were added. Filters were hybridised overnight at 42°C and then sequentially washed in 2-fold SSPE with 0.1% SDS at 42°C for 15 min, SSPE with 0.1% SDS at 65°C for 30 min, and finally 0.1-fold SSPE with 0.1% SDS at room temperature for 15 min. Specifically bound probe was visualized by autoradiography and quan- tified with a densitometer (model 300A; Molecular Dynamics, Sunnyvale, CA, U.S.A.). Relative mRNA levels were normalised by comparison with actin mRNA on the same filters. The densitometric ratio was calculated by the intensity of specific mRNAs divided by the intensity of actin mRNA.

Complementary DNA probes of ALP, OP, MGP, and pro-al(1) collagen were nick translated with ["PI a-dCTP to a specific activity of 1 x lo9 dpmlyg DNA according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany). The full length 2.4 kb rat cDNA for ALP (38) was a gift from Dr. G. Rodan (Merck, Sharp and Dohme Re- search Laboratories, West Point, PA, U.S.A.); human cDNA for OP was obtained from Dr. L. Fisher (Na- tional Institute of Dental Research, National Insti- tutes of Health, Bethesda, MD, U.S.A.); cDNA for rat MGP, from Dr. P. Price (University of San Diego, La Jolla, CA, U.S.A.); and cDNA for rat pro-al(1) collagen, from Dr. J. Bateman (Royal Children's Hospital, Melbourne, Victoria, Australia).

RESULTS

Cell Growth in Monolayer Culture

Independent treatment of UMR-201-10B cells with RA and calcitriol at maximally effective concentrations resulted in significant growth inhibition of 33 and 25% of the control, respectively (Fig. 1). AA (50 pg/ml) by itself did not result in a significant reduction of growth. However, when it was added to

J Orthop Res, Vol. 11, No. 5, 1993


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RA+CAL

CAL+AA,, i Treatment P ro toco l

FIG. 1. Effects of retinoic acid (RA), calcitriol (CAL), and ascorbic acid (AA,,) on the growth of UMR-201-1 OB in monolayer culture. UMR-201-1 OB cells were subcultured into 9.6 cm2 six-well plates at a density of 5 x l o 4 cells/well in alpha-modified Minimal Essential Medium (a-MEM; Flow Lab- oratories) containing 10% fetal bovine serum (FBS) until approximately 80% confluent; the medium then was changed to a-MEM with 2% A- FBS. The cultures were maintained at 37°C in a 5% COP air environment. Treatment was maintained for 48 h before replicate wells were harvested and counted. Samples are the mean of triplicate wells. The experiments were performed three times with similar results, and a representative result is shown. *p < 0.005, and **p < 0.001; p values were measured against the controls (C).

RA or calcitriol, there was significant enhancement of the growth inhibitory effects to 42 and 37% of the control, respectively. The combination of RA and calcitriol resulted in growth inhibition of 65%.

ALP Specific Activity

Treatment of UMR-201-10B cells with RA or calcitriol resulted in a time-dependent induction of ALP activity (Fig. 2); M RA induced ALP activity after 24 h, reaching a peak after 7 days, and treatment with lo-* M calcitriol resulted in a smaller induction of ALP activity that was evident only after 72 h and which also peaked at 7 days. Following treatment with RA and calcitriol together, ALP activity was twice that induced by RA alone after 48 h. This enhanced ALP activity occurred at least 24 h before any significant induction of ALP activity by calcitriol alone. This was a consistent finding in repeated experiments.

Treatment of UMR-201-10B cells with 50 pg/ml

AA alone did not result in any induction of ALP activity (Fig. 3), nor did higher concentrations of AA (data not shown). Combined treatment with RA and AA, however, resulted in 50% enhancement of the RA-induced ALP activity at 48 h. This was a consistent observation in repeated experiments. The result of combined treatment of UMR-201-10B cells with calcitriol and AA is seen in Fig. 4, which shows an inhibition of calcitriol-induced ALP activity during 7 days of combined treatment.

Expression of Specific mRNAs

The steady state expression of mRNA for pro a- 1(I) collagen, ALP, OP, and MGP after 7 days of treatment with the various factors was normalised against actin. Representative Northern hybridisation blots of repeated experiments are shown in Figs. 5 and 6.

Pro-a1 ( I ) Collagen

UMR-201 -lOB cells constitutively expressed the mRNA for pro-al(1) collagen on probing with a

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0 0 - 1 0 1 2 3 4 5 6 7 8 9

D u r a t i o n of T r e a t m e n t (Days)

FIG. 2. Time-dependent response of alkaline phosphatase (ALP) specific activity of UMR-201-10B cells to treatment with retinoic acid (V), calcitriol (O) , and a combination of the two (V). Cells were subcultured into 9.6 cmz six-well plates, as described in the Materials and Methods section, with a seeding density of 5 x l o 4 cells per well. The results shown are those of a representative experiment that was performed three times. The points are the mean of triplicate wells, and the bars are the SEM. Under control conditions, ALP activity is not detectable in UMR-201-1OB cells.


642 P. F: M . CHOONG ET AL.

1.6 kb Pstl insert of rat pro-al(1) collagen cDNA (15) (Fig. 5) . The steady state levels of mRNA for p r o d (I) collagen mRNA were increased following independent treatment with RA and AA. Treatment with RA resulted in a 3-fold increase in pro-al(1) collagen mRNA. Calcitriol did not alter the steady state level of pro-al(1) collagen mRNA. In comparison with the control, combined treatment with RA and AA increased the steady state level of pro-al(1) collagen mRNA by approximately 5-fold. When used

- 1 0 1 2 3 4 5 6 7 8 9 Durat ion of Treatment (Days)

FIG. 3. Time-dependent response of alkaline phosphatase specific activity of UMR-201-10B cells to treatment with retinoic acid (M), ascorbic acid (a), and a combination of the two (A). Cells were subcultured into 9.6 cm2 six-well plates, as described in the Materials and Methods section, and with a seeding density of 5 x lo4 cells per well. The results shown are those of a representative experiment that was performed three times. The points are the mean of triplicate wells, and the bars are the SEM.

concurrently, calcitriol had an inhibitory effect on RA and AA-induced expression of pro-al(1) collagen mRNA, with reductions in steady state levels of 53 and 45%, respectively.

ALP

UMR-201-1OB cells do not constitutively express the 2.4 kb mRNA for ALP (29). The addition of RA resulted in significant induction of ALP mRNA expression as shown previously (41) (Fig. 5). By comparison, calcitriol-induced ALP expression was only 27% of that shown with RA treatment alone. The

combined treatment of RA and calcitriol, however, resulted in a level of mRNA expression almost 1.5 times greater than that with RA alone. Treatment with AA alone did not cause a significant induction of ALP mRNA. When AA was added to calcitriol, however, there was an approximate 30% decrease in the calcitriol-induced ALP steady state level of mRNA.

OP

UMR-201-1OB cells constitutively expressed the 1.6 kb mRNA for OP (40) (Fig. 6). The addition of either RA or calcitriol resulted in 4.6 and 10.4-fold increases in steady state levels of OP mRNA, respectively. RA and calcitriol interacted synergistically to cause a 30-fold increase in the steady state level of OP mRNA. AA alone had no significant effect on the steady state OP mRNA level; however, when it was added to RA, it slightly attenuated the RA- induced rise in OP mRNA expression. In contrast, the combination of AA and calcitriol resulted in a significant (17-fold) enhancement in the steady state OP mRNA level.

0 1 2 5 4 5 6 7 8 9

Duration of t reatment (Days)

FIG. 4. Time-dependent response of alkaline phosphatase specific activity of UMR-201-1OB cells to treatment with calci- trio1 (M), ascorbic acid (a), and a combination of the two (A). Cells were subcultured into 9.6 cm' six-well plates, as described in the Materials and Methods section, with a seeding density of 5 x lo4 cells per well. The results shown are those of a representative experiment that was performed three times. The points are the mean of triplicate wells, and the bars are the SEM.



FIG. 5. Northern blot analyses of mRNA for pro-a1 ( I ) collagen and alkaline phosphatase (ALP) in UMR-201-1 OB cells. Treatments are indicated at the top of each lane. Total RNA (1 5 pg) was loaded in each lane. A filter was probed with a 2.4 kb rat cDNA for ALP, washed, and reprobed with a cDNA for chicken actin. A second filter was probed with cDNA for chicken actin then washed and reprobed with the insert for pro-a1 (I) collagen. Each lane represents cells treated for 1 week, with changes of medium on days 2, 4, and 6. The arrows point to the 28 and 18 S ribosomal bands. Relative densitornetric measurements are indicated beneath each lane. Variations in actin mRNA represent variations in lane loading of total mRNA as indicated by staining with ethidium bromide. RA = cells treated with 1 0-6 M retinoic acid. CAL = cells treated with 1 O-@ M calcitriol, and AA = cells treated with 50 pg/ml of ascorbic acid.

MGP

Treatment with either RA or calcitriol for 7 days induced the expression of a 700 bp mRNA (14) for MGP (Fig. 6). The combined treatment of cells with RA and calcitriol resulted in a profound augmenta- tion of the steady state level of MGP mRNA compared with treatment with RA or calcitriol alone. The addition of AA to either RA or calcitriol re-

sulted in an approximately 6-fold and 3-fold rise in MGP mRNA expression, respectively, although AA by itself had no effect on its expression.

DISCUSSION

These results indicate that RA, calcitriol, and AA have significant pleiotropic influences on the irnmor-

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644 P. E M . CHOONG ET AL.

FIG. 6. Northern blot analysis of mRNA for matrix-Gla protein (MGP) in UMR-201-106 cells. Treatments are indicated at the top of each lane. Total RNA (15 pg) was loaded in each lane. A filter was probed with a 1.6 kb mRNA for osteopontin, washed, reprobed with a plasmid containing a 700 bp rat MGP cDNA and finally washed again and reprobed with a cDNA for chicken actin. The arrow points to the 18 S band. Each lane represents cells treated for 1 week, with changes of medium on days 2, 4, and 6. Densitornetric measurements relative to actin are indicated beneath each lane. Variations in actin mRNA represent variations in lane loading of total mRNA as indicated by staining with ethidium bromide. RA = cells treated with 1 0-6 M retinoic acid, CAL = cells treated with 1 0-8 M calcitriol, and AA = cells treated with 50 pg/ml of ascorbic acid.

talized rat preosteoblast clonal cell line, UMR-201- ZOB, with effects on growth, ALP activity, and the expression of specific mRNAs.

The anti-proliferative effect of calcitriol found in UMR-201-1OB cells is consistent with other reports of such effects of calcitriol on rat calvarial osteoblasts (32) and mouse bone cells (6). The inhibition of proliferation and induction of differentiated osteoblast function by RA has been described previously (28,29,41). Although AA did not demonstrate an inhibitory action on its own, the increased inhibition of cell growth when AA was added to RA or calcitriol suggests an active modulatory role of AA.

ALP activity is widely accepted as a biochemical marker of differentiated osteoblast function (4,26, 27). UMR-201-1OB cells do not constitutively express ALP activity, which reflects their immature stage of differentiation in the osteoblast lineage. Treatment with RA resulted in significant induction of ALP activity, as previously reported (30,41), whereas calcitriol induced ALP activity after a lag period of 72 h and to a much lesser extent. A significant enhancement of ALP activity was seen after 48 h of combined treatment with RA and calcitriol, at a time when calcitriol alone had not yet induced a rise in ALP activity. The synergism observed after



combined treatment with the steroid hormones RA and calcitriol may be explained, in part, by the cross recognition of their palindromic hormone receptor elements, which share a certain homology in the DNA binding domain (1,2,39). In this context, Schule et al. (34) reported that the receptors for calcitriol and RA recognise a common response element in the human osteocalcin gene. Furthermore, it is con- ceivable that the juxtaposed human response elements may interact with each other in a manner such that the stimulation of one may modify the response of the other. Finally, it is known that RA induces the formation of calcitriol receptors in certain clonal cell lines (7,18). Such an effect may further explain the enhancement shown in this study.

Treatment with AA did not induce ALP activity in UMR-201-10B cells. It did, however, cause a 50% enhancement of RA-induced ALP activity and an inhibition of calcitriol-induced ALP activity. The mechanism for these effects remains ill defined. One possible explanation may be the indirect result of alterations in the regulation of extracellular matrix feedback under the influence of AA, through its central role in collagen synthesis. Our results lend support to this concept, which has been alluded to by studies on skin fibroblasts (22,23,35) as well as mu- rine osteoblasts (20). The changes in ALP activity correlated with alterations observed in steady state levels of ALP mRNA.

RA is a potent differentiation-promoting agent, and this effect is clearly demonstrated by the significant induction of pro-al(1) collagen, ALP, OP mRNA, and, to a lesser degree, MGP. Calcitriol dis- played an action similar to that of RA in enhancing the expression of OP, ALP, and MGP mRNAs but not pro-al(1) collagen mRNA. Northern blot analyses of combined treatment with RA and calcitriol demonstrated a significant enhancement of mRNA for ALP, with the greatest effect observed on the matrix proteins OP and MGP: increases of almost 30 and 35-fold were observed. Cotreatment of cells with calcitriol and RA resulted in a significant reduction in RA-induced steady state level of pro-al(1) collagen mRNA. Possible explanations for such significant interactions already have been discussed.

Of the various mRNAs examined in this study, the greatest influence by AA alone on the regulation of gene expression of bone extracellular matrix components was through an increase in the steady state level of pro-al(1) collagen mRNA. This suggests that the major influence of AA on bone extracellular

matrix is through its regulation of collagen synthesis. When AA was combined with either calcitriol or RA, it was obvious that interactions occurred that resulted in the modulation of the actions of individual factors. For example, cotreatment with AA and calcitriol resulted in a significant enhancement of the steady state levels of OP and MGP mRNA. On the other hand, this combination resulted in a 45% reduction in the steady state level of pro-al(1) collagen mRNA seen with AA alone. In this context, calcitriol has been shown to decrease the steady state levels of pro-al(1) collagen mRNA in ROS 1712.8 rat osteosarcoma cells (21). Treatment with AA and calcitriol also resulted in a decrease in the calcitriol- induced steady state level of ALP mRNA, which correlated with the results of assays for ALP activity. When AA was combined with RA, the steady state levels of RA-induced ALP mRNA were enhanced, which was consistent with the effect of AA on RA- induced ALP activity. The combination of AA and RA also produced variable effects on genes reg- ulating extracellular matrix proteins. Specifically, RA-induced OP mRNA steady state levels were attenuated, whereas significant enhancements of RA-induced MGP and pro-al(1) collagen mRNA expression were observed. Interestingly, OP mRNA, which contains the RGD adhesion peptide, was reduced, which perhaps suggests that AA may man- ifest part of its action through alterations in cell matrix adhesion mechanisms. For example, Harada et al. (20), using synthetic analogs containing the RGD peptide, showed a competitive modification of AA-induced phenotypic expression in MC 3T3- El cells.

The present study demonstrated effects of AA and of RA combined with calcitriol on mRNA steady state levels that have not been described previously. The molecular actions of AA obviously are complex and currently are being investigated with particular emphasis on its effects on gene transcriptional rates and mRNA stability and other post-transcriptional mechanisms whereby AA-induced matrix constitu- ents can influence cell responses.

In conclusion, RA, calcitriol, and AA are potent inducers of osteoblast differentiation and important regulators of differentiated osteoblast function. The novel interactions among all three factors highlight the complex nature of these interactions with possible roles in genomic control of the expression of the osteoblast phenotype as well as through extracellular matrix feedback mechanisms.


646 P. l? M . CHOONG ET AL.

Acknowledgment: The authors would like to acknow- ledge the support of a grant from The Royal Australasian College of Surgeons. The authors would also like to ac- knowledge Dr. Hong Zhou for her technical assistance.

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Effects of ascorbic acid, calcitriol, and retinoic acid on the differentiation of preosteoblasts

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