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Endocrinology Vol. 143, No. 7 2775-2786
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

1{alpha},25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3 Modulate Growth Plate Chondrocyte Physiology via Protein Kinase C-Dependent Phosphorylation of Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase

Z. Schwartz, H. Ehland, V. L. Sylvia, D. Larsson, R. R. Hardin, V. Bingham, D. Lopez, D. D. Dean and B. D. Boyan

Departments of Orthopedics, Periodontics, and Biochemistry, University of Texas Health Science Center (Z.S., H.E., V.L.S., V.B., R.R.H., D.L., D.D.D., B.D.B.), San Antonio, Texas 78229; Department of Periodontics, Hebrew University Hadassah (Z.S.), Jerusalem, Israel 91-010; Wilford Hall Medical Center (H.E.), Lackland Air Force Base, Texas 78236; and Department of Food and Nutrition Sciences, Utah State University (D.L.), Logan, Utah 84322

Address all correspondence and requests for reprints to: Barbara D. Boyan, Ph.D., Department of Orthopedics, MSC 7774, University of Texas Health Science, Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: . boyanb{at}uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Membrane-mediated increases in protein kinase C (PKC) activity and PKC-dependent physiological responses of growth plate chondrocytes to vitamin D metabolites depend on the state of endochondral maturation; 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25-(OH)2D3] regulates growth zone (GC) cells, whereas 24R,25-(OH)2D3 regulates resting zone (RC) cells. Different mechanisms, including protein kinase A signaling, mediate the effects of 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 on PKC, suggesting that different mechanisms may also regulate any MAPK involvement in the physiological responses. This study used confluent cultures of rat costochondral chondrocytes as a model. 1{alpha},25-(OH)2D3 stimulated MAPK specific activity in GC in a time- and dose-dependent manner, evident within 9 min. 24R,25-(OH)2D3 stimulated MAPK in RC; increases were dose dependent, occurred after 9 min, and were greatest at 90 min. In both cells the effect was due to ERK1/2 activation (p42 > p44 in GC; p42 = p44 in RC). MAPK activation was dependent on PKC, but not protein kinase A. The effect of 1{alpha},25-(OH)2D3 required phospholipase C, and the effect of 24R,25-(OH)2D3 required phospholipase D. Inhibition of cyclooxygenase activity reduced the effect of 1{alpha},25-(OH)2D3 on MAPK in GC and enhanced the effect of 24R,25-(OH)2D3 in RC. Based on MAPK inhibition with PD98059, ERK1/2 MAPK mediated the effect of 24R,25-(OH)2D3 on [3H]thymidine incorporation and [35S]sulfate incorporation by RC, but only partially mediated the effect of 1{alpha},25-(OH)2D3 on GC. ERK1/2 was not involved in the regulation of alkaline phosphatase specific activity by either metabolite. This paper supports the hypothesis that 1{alpha},25-(OH)2D3 regulates the physiology of GC via rapid membrane-mediated signaling pathways, and some, but not all, of the response to 1{alpha},25-(OH)2D3 is via the ERK family of MAPKs. In contrast, 24R,25-(OH)2D3 exerts its effects on RC via PKC-dependent MAPK. Whereas 1{alpha},25-(OH)2D3 increases MAPK activity via phospholipase C and increased prostaglandin production, 24R,25-(OH)2D3 increases MAPK via phospholipase D and decreased prostaglandin production. The cell specificity, metabolite stereospecificity, and the dependence on PKC argue for the participation of membrane receptors for 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 in the regulation of ERK1/2 in the growth plate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SECOSTEROID, 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25-(OH)2D3], regulates growth plate chondrocytes via traditional nuclear receptor-mediated mechanisms (1) as well as through membrane-associated mechanisms (2, 3, 4). Rapid membrane responses to 1{alpha},25-(OH)2D3 as well as membrane receptors for 1{alpha},25-(OH)2D3 (1,25-mVDR) have been identified in a number of cell types (5, 6, 7, 8, 9, 10, 11, 12), and activation of these membrane receptors modulates physiological responses to the vitamin D metabolite (5, 13). Studies using analogs of 1,25-(OH)2D3 that exhibit very low binding affinity for the nuclear vitamin D receptor (1,25-nVDR) provide further evidence that 1{alpha},25-(OH)2D3 elicits cell responses by mechanisms other than the traditional vitamin D receptor. These analogs cause the same physiological responses in growth zone chondrocytes as 1{alpha},25-(OH)2D3, and these effects are also blocked by antibodies to the 1,25-mVDR (5, 14). In addition, some analogs specifically block the membrane- mediated effects of 1{alpha},25-(OH)2D3 (15). Similarly, resting zone cells (RC) exhibit rapid responses to 24R,25-(OH)2D3, and the presence of a membrane receptor, distinct from that of the 1,25-mVDR, has been shown in growth plate chondrocytes and osteoblasts (5, 11).

Nuclear receptors for 24R,25-(OH)2D3 have not yet been identified biochemically, although growth plate chondrocytes exhibit specific binding sites based on autoradiography (16). The physiological responses of RC to 24R,25-(OH)2D3 can be mimicked by an analog of 1,25-(OH)2D3 that has been modified on the A ring and C,D side-chain and has low affinity for the 1,25-nVDR, and these effects are not blocked by antibodies to the 1,25-mVDR (14). These observations suggest that the response to 24R,25-(OH)2D3 is mediated by a 24,25-mVDR and not by either a traditional nuclear vitamin D receptor for either metabolite or the membrane receptor for 1{alpha},25-(OH)2D3.

Activation of membrane receptors may lead to gene expression through a number of signaling pathways. This is a relatively new concept for lipophilic hormones such as the vitamin D metabolites [see Nemere and Farach-Carson (17) and Boyan et al. (3) for reviews]. Membrane receptors for 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 both mediate target cell response through activation of protein kinase C{alpha} (PKC{alpha}) (18, 19), but different mechanisms are involved. In growth zone cartilage cells (18) and skeletal muscle cells (20, 21), PKC is stimulated by 1{alpha},25-(OH)2D3 via activation of phosphatidylinositol-specific phospholipase C (PLC), resulting in the formation of diacylglycerol and inositol-1,4,5-trisphosphate (22). 1{alpha},25-(OH)2D3 also activates phospholipase A2 (23, 24), resulting in the release of arachidonic acid (25, 26), which then serves as a substrate for cyclooxygenase-1 (27). Prostaglandin (PG) production is increased (28), and PGE2 participates in the mechanism by acting on its EP1 receptor to produce activated Gq and increase PKC activity (29). 1{alpha},25-(OH)2D3 has no effect on PKC in RC. 1{alpha},25-(OH)2D3 also stimulates protein kinase A (PKA) in muscle cells (30, 31) via G protein phosphorylation. In contrast, 24R,25-(OH)2D3 increases PKC via phospholipase D (PLD)-dependent production of diacylglycerol (32). There is a rapid inhibition of phospholipase A2 activity (25), reducing the amount of arachidonic acid available for cyclooxygenase-1, and ultimately causing a reduction in the production of PG (28). This has a stimulatory effect on PKC, however, because the PKA pathway is inhibitory of the 24R,25-(OH)2D3 effect in these cells (33). 24R,25-(OH)2D3 has no effect on PKC in growth zone cells (GC).

Inhibition of PKC blocks many of the physiological responses of GC to 1{alpha},25-(OH)2D3 and of RC to 24R,25-(OH)2D3 (3), indicating that the pathways initiated by activation of the enzyme have genomic consequences. This is true for PKA-dependent signaling as well (29, 33). Signal transduction pathways mediated by PKC and PKA converge at the activation of MAPK in many cells (15, 34, 35). This is of particular significance because MAPK phosphorylates transcription factors, such as activating protein-1 (36), thereby modulating gene expression. Whether this is the case for chondrocytes in response to these vitamin D metabolites is not known.

There are three major families of MAPK: the ERK family, the c-Jun N-terminal kinase (JNK)/stretch-activated MAPKs, and p38 (34). The ERK family consists of two proteins, p42 (ERK2) and p44 (ERK1), that are activated by phosphorylation on tyrosine residues by MAPK kinase (MEK) (37). Once activated, ERK1/2 then act as serine kinases (34). Recent reports indicate that ERK MAPKs may mediate the effects of 1{alpha},25-(OH)2D3. Inhibition of ERK1/2 activity with PD98059 blocks the effects of 1{alpha},25-(OH)2D3 on MG63 cells, including the synergistic increases in alkaline phosphatase activity observed when these cells are cultured on titanium surfaces with rough microtopographies (38). ERK1/2 are involved in regulating aggrecan expression in bovine articular chondrocytes (39), as is alkaline phosphatase in osteoblasts (40). However, it is not known whether cell membrane-mediated physiological responses to 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 are via ERK1/2 or only a subset of responses.

Recent studies suggest that increased MAPK activity in response to 1{alpha},25-(OH)2D3 does not require new enzyme expression, but is due to increased phosphorylation of existing ERK1/2 (15, 35). Moreover, the effect is mediated by membrane-associated mechanisms. In NB4 cells, p42 phosphorylation is increased by 6-s-cis-locked analogs of 1{alpha},25-(OH)2D3, but not by 6-s-trans-locked analogs (15), indicating that the secosteroid must be in a 6-s-cis conformation. 6-s-cis-locked analogs activate rapid responses of NB4 cells, intestinal epithelial cells, and osteoblasts and exhibit very low affinity for the 1,25-nVDR (15, 41), supporting the hypotheses that 1{alpha},25-(OH)2D3 regulates MAPK via a membrane receptor. Similarly, 1ß,25-(OH)2D3, which is a specific antagonist of transcaltachia induced by 1{alpha},25-(OH)2D3 (42), also reduces 1{alpha},25-(OH)2D3-dependent p42 phosphorylation. In muscle cells, MAPK activity and ERK1/2 phosphorylation in response to 1{alpha},25-(OH)2D3 are dependent on PKC stimulation (35), suggesting that MAPK may be regulated by the 1,25-mVDR signaling pathway. It is not known whether MAPK is also regulated by a 24,25-mVDR, nor is it known whether MAPK is regulated by the same mechanisms that mediate the rapid PKC response to 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3.

These observations suggest that 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 may exert their effects on growth zone and resting zone chondrocytes at least in part through activation of the ERK family of MAPKs, but separate mechanisms are involved. The present study determined whether these vitamin D metabolites regulate ERK1/2 in their respective target cells and determined whether activation of ERK1/2 mediates the physiological response of GC and RC to 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3. We also examined the mechanisms involved in the activation of MAPK by the vitamin D metabolites, particularly focusing on the roles of PKC and PKA, since both signaling pathways can result in activation of this enzyme.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). 1ß,25-(OH)2D3 and 24S,25-(OH)2D3 were gifts from Dr. Anthony W. Norman (University of California, Riverside, CA). Ab99, a rabbit polyclonal antibody generated to the N-terminal amino acid sequence of the [3H]1,25-(OH)2D3-binding protein in the basal lateral membranes of chick intestinal epithelium (43), was a gift from Dr. Ilka Nemere (Utah State University, Logan, UT). The following chemicals were purchased from Calbiochem (San Diego, CA): PD98059 (MEK inhibitor) (44), chelerythrine and H-7 (PKC inhibitors) (45, 46), H-89 (PKA inhibitor) (47), U73122 (phosphatidylinositol-specific PLC inhibitor) (48), wortmannin (PLD inhibitor) (49), actinomycin D (transcription inhibitor), and cycloheximide (translation inhibitor). Indomethacin (cyclooxygenase inhibitor) was purchased from Sigma (St. Louis, MO). The Biotrack p42/p44 MAPK assay kit was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). DMEM was obtained from Life Technologies, Inc. (Gaithersburg, MD), and fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA). The protein content of each sample was determined using the bicinchoninic acid protein assay reagent (50) obtained from Pierce Chemical Co. (Rockford, IL). [32P]ATP, [3H]thymidine, and [35S]sulfate were obtained from NEN Life Science Products-DuPont (Boston, MA).

Chondrocyte cultures
The culture system used in this study has been described in detail previously (51). Chondrocytes were isolated from the resting zone (reserve zone) and growth zone (prehypertrophic/upper hypertrophic cell zones) of the costochondral junction of 125-g male Sprague Dawley rats and cultured in DMEM containing 10% fetal bovine serum and 50 µg/ml vitamin C in an atmosphere of 5% CO2 and 100% humidity at 37 C. Fourth passage cells were used for all experiments, as prior studies have shown retention of differential phenotype, including responses to 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3, at this number of passages (2, 3). At confluence, media were removed and replaced with experimental medium containing 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3 at the concentrations indicated below.

Regulation of MAPK by 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3
MAPK specific activity was determined using the Biotrack p42/p44 MAPK assay kit following the manufacturer’s directions. To determine whether the regulation of MAPK by 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 was metabolite specific and cell specific, cultures were treated with the different vitamin D3 metabolites. For GC, cultures in 24-well plates were treated for various time periods with 0.5 ml vehicle control (0.02% ethanol in DMEM and 10% fetal bovine serum) or experimental DMEM, 10% fetal bovine serum, plus 10-8 M 1{alpha},25-(OH)2D3, or for 9 min with 10-10, 10-9, or 10-8 M 1{alpha},25-(OH)2D3, 1ß,25-(OH)2D3, or 24R,25-(OH)2D3. For RC, cultures in 24-well plates were treated for various periods of time with 0.5 ml vehicle control (0.02% ethanol in DMEM and 10% fetal bovine serum) or experimental DMEM, 10% fetal bovine serum, and 10-7 M 24R,25-(OH)2D3, or for 90 min with 10-9, 10-8, or 10-7 M 24R,25-(OH)2D3, 24S,25-(OH)2D3, or 1{alpha},25-(OH)2D3. These concentrations were selected based on numerous previous studies in our laboratory showing that, regardless of the parameter being examined, the greatest response in GC was found in cultures treated with 10-8 M 1{alpha},25-(OH)2D3, whereas the greatest response in RC was found in cultures treated with 10-7 M 24R,25-(OH)2D3.

After the appropriate incubation period, cell layers were washed with PBS and lysed in solubilization buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40] for 30 min on ice. The cell layer lysates were assayed for protein content (50) and MAPK activity. Fifteen microliters of each experimental sample were mixed with 10 µl substrate buffer and 5 µl Mg/ATP reagent, both provided in the kit. Immediately before use, [32P]ATP was added to the Mg/ATP reagent to a final concentration of 200 µCi/ml. After a 30-min incubation at 30 C, the reaction was stopped by addition of 10 µl stop reagent, the samples were centrifuged for 15 sec at 14,000 rpm, and 30 µl were spotted onto phosphocellulose discs. The discs were washed twice with 75 mM orthophosphoric acid and once with distilled water and were counted in a liquid scintillation counter.

Western blot analysis
Chondrocytes have been shown to express ERK1/2, p38, and JNK (39, 52, 53, 54, 55, 56, 57, 58); however, this work focused only on the ERK1/2 family. To determine whether 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3 regulated the levels of ERK1/2 protein or MAPK was activated by tyrosine phosphorylation (34), cell culture lysates were examined by Western blot using specific antibodies to nonphosphorylated and phosphorylated p42/p44. Cell culture lysates were prepared from confluent, fourth passage growth zone cell cultures that had been treated for 1, 9, or 90 min with 10-8 M 1{alpha},25-(OH)2D3, or from resting zone cell cultures treated for 1, 9, 90, or 270 min with 10-7 M 24R,25-(OH)2D3 and were resolved on 10% SDS-polyacrylamide gels. Similar gels were run using cell culture lysates prepared from GC cultures treated for 9 min with 10-10, 10-9, or 10-8 M 1{alpha},25-(OH)2D3 or from RC cultures treated for 90 min with 10-9, 10-8, or 10-7 M 24R,25-(OH)2D3. Blots of the gels were probed with 1:5000 dilutions of rabbit polyclonal antibodies to phosphorylated p42/p44 or nonphosphorylated p42/p44 (Promega Corp., Madison, WI), followed by 1:5000 dilutions of alkaline phosphatase-conjugated antirabbit IgG1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The ERK1 (44 kDa) and ERK2 (42 kDa) bands were visualized using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate reagent (Sigma).

MAPK mRNA levels
To determine whether chondrocytes express genes for ERK1 and ERK2, RNA preparations from RC and GC cultures were screened by RT-PCR. Total RNA was isolated with TRIzol reagent (Life Technologies, Inc.). For sequence determination, total RNA from RC and GC were reverse transcribed with the First-Strand cDNA synthesis kit (Pharmacia Biotech, Piscataway, NJ) and sequenced by the Center for Advanced DNA Technologies, Department of Microbiology, University of Texas Health Science Center (San Antonio, TX). For rat brain, kidney, and liver RNA samples, 1 µg total RNA (purchased from Ambion, Inc., Austin, TX) was used in reverse transcriptase reactions. Rat primer sequences were generated from rat sequences (available in GenBank) and based on primer sequences shown to amplify rat ERK cDNAs. The sets of ERK primers were: ERK1 sense, 5'-GAT TGC TGA CCC TGA GCA C-3'; and ERK1 antisense, 5'-GGG GGC CTC TGG TGC C-3'; ERK2 sense, 5'-GCC CGG AGA TGG TCC GC-3'; and ERK2 antisense, 5'-ATG GTC TGG ATC TGC AAC A-3'. The expected product sizes were 570 bp (ERK1) and 506 bp (ERK2). Rat brain RNA served as positive control template for ERK1 and ERK2. Sequences were confirmed by direct comparison with published rat ERK1 and ERK2 sequences available in GenBank [ERK1, accession no. M61177 (59); ERK2, accession no. M64300 (60)].

Northern blot analysis
To quantitate the effects of 1{alpha},25-(OH)2D3 on mRNA levels for ERK1 and ERK2, we performed Northern blot analysis. Total RNA for untreated and 1{alpha},25-(OH)2D3-treated growth zone chondrocytes as well as from untreated and 24R,25-(OH)2D3-treated RC was isolated with TRIzol. Total RNA was quantitated spectrophotometrically, separated on a 1% denaturing agarose gel, and transferred to a positively charged nylon membrane (Ambion, Inc.) with the Turboblotter System (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). Northern blots were hybridized with ERK1, ERK2, and glyceraldehyde-3-phosphate dehydrogenase-strippable 32P-labeled anticoding RNA probes using the NorthernMax Kit (Ambion, Inc.). ERK1 and ERK2 DNA templates were synthesized from sequenced RT-PCR products amplified with modified antisense primers with the T7 promoter sequence, 5'-TAA TAG GAC TCA CTA TAG GGA GG-3', attached to the 5'-end of the antisense primers. Anticoding RNA probes were synthesized with the Strip-EZ T7 Kit (Ambion, Inc.). Northern blots were analyzed with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Requirement for gene expression and protein synthesis
To determine whether new gene expression or protein synthesis is required for 1{alpha},25-(OH)2D3- and 24R,25-(OH)2D3-dependent stimulation of MAPK activity, growth zone and resting zone chondrocytes were treated with 1, 10, or 100 µM actinomycin D to block transcription or with 1, 10, or 100 µM cycloheximide to block translation (61). These doses were selected based on previous studies using the costochondral growth plate chondrocyte model (18, 61).

PKC and PKA
1{alpha},25-(OH)2D3 mediates its effects on GC via the PKC and PKA signaling pathways (62). These pathways also mediate the effects of 24R,25-(OH)2D3 on RC (4). To determine whether activation of MAPK involved one or both mechanisms, specific inhibitors of each pathway were used. Two different inhibitors were used to inhibit PKC, chelerythrine (0.1, 1, or 10 µM) (45) and H-7 (0.1 or 1 µM) (46). Growth zone chondrocyte cultures were incubated for 9 min in control medium or medium containing 10-8 M 1{alpha},25-(OH)2D3 with or without chelerythrine or H-7, resting zone cultures were incubated for 90 min in control medium or medium containing 10-7 M 24R,25-(OH)2D3 with or without chelerythrine or H-7, and MAPK activity was determined. The involvement of PKA was assessed in a similar manner using the PKA inhibitor H-89 (0.1 or 1 µM) (47).

PLC
PLC mediates the effects of 1{alpha},25-(OH)2D3 on PKC in growth zone cell cultures (22), but does not mediate the effects of 24R,25-(OH)2D3 on RC (63). Its involvement in the effect of vitamin D3 metabolites on MAPK was assessed by using U73122, an inhibitor of phosphatidylinositol specific (PI)-PLC (48), at 0.1, 1.0, or 10 µM. Growth zone chondrocytes were incubated for 9 min in control medium or medium containing 10-8 M 1{alpha},25-(OH)2D3 with or without U73122, and RC cultures were incubated for 90 min in control medium or medium containing 10-7 M 24R,25-(OH)2D3 with or without U73122.

PLD
PLD mediates the membrane effects of 24R,25-(OH)2D3 on PKC activity of RC, but not GC (63). Its involvement in the effects of vitamin D3 metabolites on MAPK activity was assessed using wortmannin, an inhibitor of PLD (49, 64). GC cultures were incubated for 9 min in control medium or medium containing 10-8 M 1{alpha},25-(OH)2D3 with or without 0.1, 1.0, or 10 µM wortmannin. RC were incubated for 90 min in control medium or medium containing 10-7 M 24R,25-(OH)2D3 with or without the same concentrations of wortmannin.

Cyclooxygenases
Inhibition of PG synthesis blocks the effects of 1{alpha},25-(OH)2D3 on PKC in GC and augments the effects of 24R,25-(OH)2D3 in RC (27, 65). Involvement of PGs in the regulation of MAPK by these metabolites was assessed using indomethacin, a general inhibitor of cyclooxygenases. GC were incubated for 9 min in control medium or medium containing 10-8 M 1{alpha},25-(OH)2D3 with or without 10-8 or 10-7 M indomethacin. RC were incubated for 90 min in control medium or medium containing 10-7 M 24R,25-(OH)2D3 with or without 10-8 M or 10-7 M indomethacin.

Role of MAPK in mediating the physiologic response to vitamin D3 metabolites
Vitamin D3 metabolites inhibit [3H]thymidine incorporation by resting zone and growth zone chondrocytes (66). To determine whether this response is mediated by MAPK, DNA synthesis was estimated by measuring [3H]thymidine incorporation into trichloroacetic acid-insoluble cell precipitates as described previously (66). Quiescence was induced by incubating confluent cultures for 48 h in DMEM containing 1% fetal bovine serum. The medium was then replaced with DMEM containing 1% fetal bovine serum alone (control), 10-10–10-8 M 1{alpha},25-(OH)2D3 for GC, or 10-9–10-7 M 24R,25-(OH)2D3 for RC in the presence or absence of 1, 10, or 100 µM PD98059 for 24 h. PD98059 prevents activation of MAPK by inhibiting the action of MEK, which is the enzyme that phosphorylates ERK1/2 (44). Two hours before harvest, [3H]thymidine was added.

Alkaline phosphatase specific activity
1{alpha},25-(OH)2D3 stimulates alkaline phosphatase specific activity in GC cultures, whereas 24R,25-(OH)2D3 increases this enzyme activity in RC cultures (67). To determine whether this effect was mediated by MAPK, confluent cultures were treated with medium containing vehicle alone, 10-10–10-8 M 1{alpha},25-(OH)2D3 for GC, or 10-9–10-7 M 24R,25-(OH)2D3 for RC in the presence or absence of 1, 10, or 100 µM PD98059 (MEK inhibitor) for 24 h. Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, alkaline (EC 3.1.3.1)] specific activity was measured in cell layer lysates as a function of release of para-nitrophenol from para-nitrophenylphosphate at pH 10.2, as described previously (67, 68, 69).

Proteoglycan sulfation
1{alpha},25-(OH)2D3 causes an increase in [35S]sulfate incorporation in GC cultures, whereas 24R,25-(OH)2D3 regulates proteoglycan sulfation in cultures of RC (70). To determine whether this is mediated by MAPK, proteoglycan synthesis was assessed by measuring [35S]sulfate incorporation by confluent cultures as described by us previously (71, 72). At confluence, fresh medium containing vehicle alone, 10-10–10-8 M 1{alpha},25-(OH)2D3 for GC cultures, or 10-9–10-7 M 24R,25-(OH)2D3 for RC cultures in the presence or absence of 1, 10, or 100 µM PD98059 (MEK inhibitor) for 24 h. Four hours before harvest, 50 µl DMEM containing 18 µCi/ml [35S]sulfate and 0.814 mM carrier sulfate were added to each culture. At harvest, the conditioned media were removed, the cell layers (cells and matrix) were collected, and the amount of [35S]sulfate incorporated was determined as a function of cell layer protein.

Statistical management of data
For each experiment, each value represents the mean ± SEM of the cell layers of six individual independent cultures. Significance between groups was determined by ANOVA. Bonferroni’s modification of the t test was used for post hoc testing. P < 0.05 was considered significant. Each experiment was repeated two or more times to ensure the validity of the data. The data presented are from a single representative experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GC and RC both expressed mRNA for ERK1/2 (p42/p44) based on RT-PCR (data not shown) and Northern blot analysis (Fig. 1Go), and the amount of expression was similar in both types of cells.



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  		Figure 1. Effects of 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 on mRNA levels for p44 and p42 MAPK in rat costochondral growth plate chondrocytes. Confluent cultures of GC were treated for 90 min with 10-10–10-8 M 1{alpha},25-(OH)2D3 and confluent RC cultures were treated with 10-9–10-7 M 24R,25-(OH)2D3 for the same length of time. RNA was prepared from the cells and analyzed by Northern blot using DNA probes as described in Materials and Methods. The relative intensities of the bands for p44 or p42 were compared with the intensity of GAPDH controls run simultaneously in each lane.

 
There was a time-dependent decrease in MAPK activity in controls of the RC cultures (Fig. 2Go). This was due to the experimental design. The addition of fresh medium to these cultures at time zero increased enzyme activity. As bioactive agents in the medium were depleted, the medium-dependent stimulation was reduced. This effect was not seen in the GC cultures. 1{alpha},25-(OH)2D3 regulated MAPK specific activity of GC in a time-dependent manner (Fig. 2AGo). Enzyme activity was increased by 10-8 M within 9 min, and the greatest activity was observed at 90 min. By 180 min, MAPK activity had returned to control levels. The effect of 1{alpha},25-(OH)2D3 was dose dependent (Fig. 2BGo). Increased activity was noted in cultures treated with 10-10 M, and the highest levels of activity were seen in cultures treated with 10-8 M 1{alpha},25-(OH)2D3. RC responded to 24R,25-(OH)2D3 in a time-dependent manner as well, but the effect on MAPK did not occur until after 9 min (Fig. 2CGo). Peak activity was seen at 90 min and remained elevated over control cultures through 270 min. The effect of 24R,25-(OH)2D3 was dose dependent, with greatest activation at 10-7 M (Fig. 2DGo). These observations were consistent, as indicated by analysis of treatment/control ratios of five independent experiments (data not shown).



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  		Figure 2. Effect of vitamin D3 metabolites on MAPK specific activity of growth zone and resting zone chondrocyte cultures. Confluent, GC were treated for 9–720 min with 10-8 M 1{alpha},25-(OH)2D3 (A) or with 10-10–10-8 M 1{alpha},25-(OH)2D3 for 9 min (B). Alternatively, confluent RC were treated for the same time intervals, but with 10-7 M 24R,25-(OH)2D3 (C) or with 10-9–10-7 M 24R,25-(OH)2D3 for 90 min (D). In all cases, at harvest MAPK specific activity was measured in cell layer lysates as described in Materials and Methods. Values are the mean ± SEM for six cultures from one experiment. The experiment was repeated four more times with nearly identical results. *, P < 0.05 vs. 9 min (A and C) or control (B and D); #, P < 0.05 vs. control for a particular treatment time (A and C), 10-10 M 1{alpha},25-(OH)2D3 (B), or 10-9 M 24R,25-(OH)2D3 (D).

 
The effects of 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 on MAPK were stereospecific and cell maturation specific (data not shown). 1{alpha},25-(OH)2D3 had no effect on MAPK activity in RC, and 24R,25-(OH)2D3 had no effect on MAPK in GC. Only 1{alpha},25-(OH)2D3 stimulated enzyme activity in GC; 1ß,25-(OH)2D3 was without effect. Similarly, only 24R,25-(OH)2D3 stimulated enzyme activity in RC; 24S,25-(OH)2D3 was without effect.

The effect of the vitamin D metabolites on MAPK activity was not due to a change in gene expression in either cell type (Fig. 1Go). No change in p44 or p42 mRNA levels were noted in Northern blots of RNA isolated from GC treated with 1{alpha},25-(OH)2D3 at any of the time points (9 and 90 min and 24 h) examined. Similarly, 24R,25-(OH)2D3 did not affect mRNA levels in RC.

Protein synthesis was also not responsible for the change in enzyme activity. Neither cycloheximide nor actinomycin D altered MAPK activity in control cultures or in cultures treated with 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3 at any of the concentrations examined (1, 10, and 100 µM; data not shown). Moreover, there were no changes in p42 or p44 protein levels in Western blots of the cell lysates (data not shown). In contrast, phosphorylation of ERK1/2 was regulated by the vitamin D metabolites in a time- and dose-dependent manner (Fig. 3Go). Phosphorylated p42 and p44 were evident in GC treated with 10-8 M 1{alpha},25-(OH)2D3 as early as 1 min, and the greatest phosphorylation was achieved at 90 min. Phosphorylation was seen in cultures treated with 10-10 M, particularly the p42 band. The greatest amount of phosphorylation was seen in cultures treated with 10-8 M 1{alpha},25-(OH)2D3. The effect of 24R,25-(OH)2D3 on the phosphorylation of MAPK in RC was similar. Phosphorylated p42/p44 was evident within 1 min and increased over time, becoming maximal at 90 min. By 270 min, there was no more evidence of phosphorylated enzyme. Phosphorylation was dose dependent, with the greatest amount in cells cultured in the presence of 10-7 M 24R,25-(OH)2D3.



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  		Figure 3. Western blot analysis of phosphorylated p42/p44 (ERK1/2) in cell layer lysates of growth zone and resting zone chondrocyte cultures. Cell layer lysates of GC cultures that had been treated for 1, 9, or 90 min with 10-8 M 1{alpha},25-(OH)2D3 (upper left panel) or for 9 min with 10-10–10-8 M 1{alpha},25-(OH)2D3 (upper right panel) were electrophoresed on SDS-polyacrylamide gels and then transferred for Western blot analysis using antibody to the phosphorylated form of p42/p44. Alternatively, cell layer lysates of RC cultures that had been treated for 1, 9, 90, or 270 min with 10-7 M 24R,25-(OH)2D3 (lower left panel) or for 90 min with 10-9–10-7 M 24R,25-(OH)2D3 (lower right panel) were electrophoresed, transferred, and analyzed using the same antibody.

 
In both GC and RC, MAPK activity was regulated by a PKC-dependent mechanism that did not involve PKA. Inhibition of PKC with chelerythrine caused a dose-dependent decrease in 1{alpha},25-(OH)2D3-stimulated MAPK in GC (Fig. 4AGo) and 24R,25-(OH)2D3-stimulated MAPK in RC (Fig. 4BGo). Chelerythrine did not alter MAPK activity in control cultures. Similar effects were found when the cultures were treated with the PKC inhibitor H-7 (data not shown). However, when the cells were treated with the PKA inhibitor H-89, there was no change in MAPK activity in the control cultures or in cultures treated with 1{alpha},25-(OH)2D3 (Fig. 4CGo) or 24R,25-(OH)2D3 (Fig. 4DGo).



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  		Figure 4. Effect of PKC inhibition and PKA inhibition on MAPK specific activity of growth zone and resting zone chondrocytes. Confluent, GC were treated for 9 min with 10-8 M 1{alpha},25-(OH)2D3 in the presence and absence of 0.1, 1, and 10 µM chelerythrine to inhibit PKC (A) or 0.1 and 1.0 µM H-89 to inhibit PKA (C). Alternatively, confluent RC were treated for 90 min with 10-7 M 24R,25-(OH)2D3 in the presence and absence of chelerythrine (B) or H-89 (D). At harvest, MAPK specific activity in cell layer lysates was measured as described in Materials and Methods. Values are the mean ± SEM for six cultures from one experiment. The experiment was repeated with nearly identical results. *, P < 0.05 vs. not treated with chelerythrine or H-89; #, P < 0.05 vs. control for a particular vitamin D3 metabolite treatment.

 
The effect of 1{alpha},25-(OH)2D3 on MAPK involved PI-PLC, but not PLD. The PI-PLC inhibitor U73122 caused a dose-dependent decrease in the MAPK activity of the treated GC cultures (Fig. 5AGo), but the PLD inhibitor wortmannin was without effect (Fig. 5BGo). In contrast, U73122 had no effect on MAPK in RC treated with 24R,25-(OH)2D3 (Fig. 5CGo), whereas activity was inhibited by wortmannin (Fig. 5DGo).



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  		Figure 5. Effects of PLC and PLD inhibitors on MAPK specific activity of growth zone and resting zone chondrocytes. Confluent, GC were treated for 9 min with 10-8 M 1{alpha},25-(OH)2D3 in the presence and absence of 0.1–10 µM U73122, a PLC inhibitor (A), or 0.1–10 µM wortmannin, a PLD inhibitor (B). Alternatively, confluent RC were treated for 90 min with 10-7 M 24R,25-(OH)2D3 in the presence and absence of 0.1–10 µM U73122, a PLC inhibitor (C), or 0.1–10 µM wortmannin, a PLD inhibitor (D). At harvest, MAPK specific activity in cell layer lysates was measured as described in Materials and Methods. Values are the mean ± SEM for six cultures from one experiment. The experiment was repeated with nearly identical results. *, P < 0.05 vs. not treated with PLC or PLD inhibitor; #, P < 0.05 vs. control for a particular vitamin D3 metabolite treatment.

 
Inhibition of cyclooxygenase activity by indomethacin affected MAPK activity in GC and RC in different ways (Table 1Go). Indomethacin reduced MAPK activity in control cultures of GC and partially reduced the increase in enzyme activity due to 1{alpha},25-(OH)2D3. In contrast, indomethacin stimulated MAPK activity in control cultures of RC to the same extent as seen in cultures treated with 10-7 M 24R,25-(OH)2D3. When RC were treated with indomethacin and 24R,25-(OH)2D3 together, MAPK activity was increased by 20% over the activity found in cultures treated with either agent alone.


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  		Table 1. Effect of indomethacin on MAPK specific activity of growth zone and resting zone chondrocytes treated with vitamin D3 metabolites

 
The ERK1/2 MAPKs mediated some, but not all, of the physiological effects of 1{alpha},25-(OH)2D3 on GC and 24R,25-(OH)2D3 on RC. ERK1/2 did not mediate the inhibitory effect of 1{alpha},25-(OH)2D3 on the proliferation of GC. Inhibition of MAPK activity with PD98059 reduced [3H]thymidine incorporation in control cultures of both GC and RC (Fig. 6Go, A and D). In GC, the effects of 1 and 10 µM PD98059 were similar to the effect of 10-8 M 1{alpha},25-(OH)2D3. At 100 µM, PD98059 caused a further reduction in [3H]thymidine incorporation over that seen in cultures treated with 1{alpha},25-(OH)2D3. In contrast, the antiproliferative effect of 24R,25-(OH)2D3 in RC was mediated by ERK1/2. Ten and 100 µM inhibitor reduced [3H]thymidine incorporation below levels seen in RC cultures treated with 24R,25-(OH)2D3 alone, and at the highest concentration, the effect of PD98059 was comparable to that of inhibitor plus 24R,25-(OH)2D3.



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  		Figure 6. Effects of MAPK inhibitor on growth plate chondrocyte proliferation, matrix synthesis, and differentiation. Confluent GC were treated for 24 h with 10-8 M 1{alpha},25-(OH)2D3 and confluent RC were treated for the same time period with 10-7 M 24R,25-(OH)2D3 in the presence and absence of 0–100 µM PD98059, a MAPK inhibitor. Cultures were labeled and harvested as described in Materials and Methods. Cell proliferation was assessed by measuring [3H]thymidine incorporation (A and D), and matrix synthesis was assessed by measuring [35S]sulfate incorporation (B and E). The effect on cell differentiation was assessed by measuring alkaline phosphatase specific activity (C and F). Values are the mean ± SEM for six cultures from one experiment. The experiment was repeated with nearly identical results. *, P < 0.05 vs. not treated with MAPK inhibitor; #, P < 0.05 vs. control for a particular vitamin D3 metabolite treatment.

 
Proteoglycan sulfation was mediated in part by ERK1/2 in GC (Fig. 6BGo). PD98059 caused a dose-dependent decrease in control cultures that was significant only at the two highest concentrations, and a similar decrease was noted in the 1{alpha},25-(OH)2D3-treated cultures. In contrast, ERK1/2 mediated the effect of 24R,25-(OH)2D3 on [35S]sulfate incorporation by RC cultures (Fig. 6EGo). The inhibitor caused a small decrease in proteoglycan sulfation in control cultures at the highest concentration used, and it blocked the 24R,25-(OH)2D3-stimulated increase in a dose-dependent manner. Alkaline phosphatase specific activity was not regulated via ERK1/2 in either control cultures or cultures treated with the appropriate vitamin D3 metabolite (Fig. 6Go, C and F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate for the first time that the ERK1/2 MAPKs are regulated by 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 in growth plate chondrocytes in a cell maturation-specific manner. 1{alpha},25-(OH)2D3 stimulates MAPK enzyme activity only in cultures of GC, and 24R,25-(OH)2D3 stimulates activity only in RC cultures. Although others have shown that 1{alpha},25-(OH)2D3 activates ERK1/2 MAPKs, this is the first demonstration that 24R,25-(OH)2D3 does so as well.

The effects of the vitamin D metabolites are due to activation via phosphorylation and not via a change in mRNA levels or protein synthesis. These results support those of Morelli et al. (35) showing that 1{alpha},25-(OH)2D3 stimulates the phosphorylation of p42 and p44 in skeletal muscle cells. In addition, our results support the observations of Song et al. (15), indicating that p42 is phosphorylated to a greater extent than p44 in response to 1{alpha},25-(OH)2D3. Further, our results show that 24R,25-(OH)2D3 increases phosphorylation of both ERKs to a similar extent, suggesting that p44 phosphorylation may involve a mechanism specific to the action of this metabolite.

The cell specificity of the responses to 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 was not due to a difference in the amount of ERK1/2 present in control cultures. No differences were noted in the levels of mRNA or protein. However, activity in the control cultures differed, with RC having approximately 3 times more specific activity than GC. This supports the observation of Oh et al. (58) that as chondrocyte differentiation increases in chick limb buds, MAPK activity decreases.

Stereospecificity argues for a receptor-mediated mechanism. It is likely that the change in MAPK activity is due to activation of membrane receptors rather than to the 1,25-nVDR for a number of reasons. First and foremost is the lack of evidence for a change in gene expression or protein synthesis, as noted above. Secondly, a nuclear receptor for 24R,25-(OH)2D3 has not yet been isolated, yet 24R,25-(OH)2D3 modulated MAPK activity and ERK1/2 phosphorylation in RC. Finally, MAPK activity in both cell types was regulated via a PKC-dependent mechanism as well as by signaling pathways previously shown to regulate PKC activity in response to the vitamin D metabolites in a cell-specific manner. 1{alpha},25-(OH)2D3 has been repeatedly shown to stimulate PKC{alpha} activity only in GC, whereas 24R,25-(OH)2D3 increases PKC{alpha} activity only in RC (18). Moreover, 1{alpha},25-(OH)2D3 increases PKC via a PLC-dependent mechanism only in GC (22), whereas 24R,25-(OH)2D3 increases PKC via a PLD-dependent mechanism only in RC (63).

The involvement of PKC in the activation of MAPK was demonstrated clearly using two separate inhibitors, chelerythrine and H-7. PKC also mediates the effects of 1{alpha},25-(OH)2D3 on MAPK in smooth muscle cells (35). Chelerythrine is commonly used to block PKC-dependent effects, but it has been reported to inhibit PKA at high concentrations (73), much higher than those used in the present study. It is unlikely that PKA plays a role in the activation of MAPK by either 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3, however, because H-89 had no effect on either enzyme activity in control cultures or in cultures treated with the vitamin D metabolites. In contrast, PKA is involved in the regulation of MAPK in response to other regulatory factors (40, 55, 57).

PGs appear to be involved in the maintenance of MAPK at basal levels in growth plate chondrocytes, as indomethacin caused a decrease in activity in control cultures of GC and an increase in activity in RC. Whether this is due to a change in ERK1/2 or another MAPK is not known. Similar effects have been observed when measuring PKC activity in growth plate chondrocytes in the presence of indomethacin (22, 32). 1{alpha},25-(OH)2D3 stimulates PG production by GC, whereas 24R,25-(OH)2D3 decreases PG production by RC, and basal production of PGE1 and PGE2 is greater in RC cultures (28), suggesting that one role of the differential response of phospholipase A2 to the vitamin D metabolites is to optimize PG levels for signal transduction via PKC and MAPK.

The effect of 1{alpha},25-(OH)2D3 on MAPK was partially blocked by indomethacin, but it is possible that this reflects the reduction in basal activity. 1{alpha},25-(OH)2D3 stimulates PKC via a mechanism that involves PG production; inhibition of cyclooxygenase-1 reduces the effect of 1{alpha},25-(OH)2D3 on PKC via PGE2-dependent activation of the E-series prostanoid-1 receptor (27, 29). As 1{alpha},25-(OH)2D3 increases MAPK via a PKC- mediated pathway, the partial reduction in enzyme activity may reflect the decrease in PKC due to indomethacin. In RC, the effect of indomethacin was comparable to that of 24R,25-(OH)2D3 alone, and it augmented the effect of this vitamin D metabolite, suggesting that PGs are involved in the mechanism in these cells as well. PKC activity is stimulated by inhibition of PG synthesis, in part because of the reduction of PKA-dependent inhibition of this signaling pathway (74).

This study supports the observation that 1{alpha},25-(OH)2D3 modulates the physiological responses of chick intestinal epithelial cells via MAPK (75). It links the membrane receptor-mediated activation of PKC to gene expression in chondrocytes. Moreover, it shows that 24R,25-(OH)2D3 can modulate gene expression via a 24,25-mVDR without invoking the need for a nuclear receptor for this vitamin D metabolite.

The results clearly demonstrate that this signaling pathway mediates some, but not all, of the physiological responses of chondrocytes to 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3. [3H]Thymidine incorporation is decreased, and alkaline phosphatase specific activity is increased in GC by 1{alpha},25-(OH)2D3 via a 1,25-mVDR and PKC (5), but ERK1/2 is not involved. Similarly, 24R,25-(OH)2D3 decreases [3H]thymidine incorporation and increases alkaline phosphatase specific activity in RC via a 24,25-mVDR and PKC (5), but, again, ERK1/2 is not involved. In contrast to our results, MAPK does mediate the effects of T3 on alkaline phosphatase in osteoblasts (40). It is unlikely that the decrease in proliferation or the increase in activity of this enzyme in response to 1{alpha},25-(OH)2D3 or 24R,25-(OH)2D3 are due only to a nongenomic mechanism, although nongenomic regulation does play a role, at least with respect to matrix vesicle enzyme activity (76). It is more likely that other MAPKs, such as JNK and p38, or other PKC-dependent signaling mechanisms are involved in the response of growth plate chondrocytes.

It is also evident that there are differences between the two maturation states in the management of the response to either metabolite via MAPK. This signaling pathway plays little, if any, role in the use of sulfate by GC in response to 1{alpha},25-(OH)2D3, although ERK MAPKs do mediate the effects of TGFß on aggrecan expression in ATDC5 cells (39). In contrast, proteoglycan synthesis by RC in response to 24R,25-(OH)2D3 is sensitive to ERK1/2 inhibition, supporting the hypothesis that the 24,25-mVDR must rely on nonnuclear vitamin D receptor-mediated mechanisms. This appears to be the case for the antiproliferative response as well.

In summary, this paper supports the hypothesis that 1{alpha},25-(OH)2D3 regulates the physiology of growth zone chondrocytes via rapid membrane-mediated signaling pathways. Some, but not all, of the response to 1{alpha},25-(OH)2D3 is via the ERK family of MAPKs, involving rapid PKC-dependent phosphorylation of p42 and, to a lesser extent, p44. This paper also shows for the first time that 24R,25-(OH)2D3 exerts its cell-specific effects on resting zone chondrocytes via PKC-dependent MAPK, affecting phosphorylation of p42 and p44 to a comparable extent. Whereas 1{alpha},25-(OH)2D3 increases MAPK activity via PLC and increased PG production, 24R,25-(OH)2D3 increases MAPK via PLD and decreased PG production. PKA is not involved in the activation of ERK1/2 in either cell type. The stereospecificity of the effects of each metabolite, the cell specificity of the effect, and the dependence on PKC argue for the participation of membrane receptors for 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 in the regulation of ERK1/2 in the growth plate.


   Acknowledgments
 
The authors thank Sandra Messier for her contributions to the preparation of the manuscript.


   Footnotes
 
This work was supported by USPHS Grants DE-05937 and DE-08603. The opinions expressed herein do not necessarily reflect the opinions of the United States Air Force.

*H.E. is a Fellow in the Air Force Institute of Technology.

Abbreviations: 1{alpha},25-(OH)2D3, 1{alpha},25-Dihydroxyvitamin D3; GC, growth zone cells; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; 1,25-mVDR, membrane receptor for 1{alpha},25-(OH)2D3; 1,25-nVDR, nuclear vitamin D receptor; PG, prostaglandin; PI, phosphatidylinositol; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; RC, resting zone cells.

Received January 22, 2002.

Accepted for publication March 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Klaus G, Von Eichel BV, May T, Hugel U, Mayer H, Ritz E, Mehls O 1994 Synergistic effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on proliferation and vitamin D receptor expression of rat growth cartilage cells. Endocrinology 135:1307–1315[Abstract]
  2. Boyan BD, Dean DD, Sylvia VL, Schwartz Z 1997 Cartilage and vitamin D: genomic and nongenomic regulation by 1,25-(OH)2D3 and 24,25-(OH)2D3. In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. San Diego: Academic Press; 395–421
  3. Boyan BD, Sylvia VL, Dean DD, Del Toro F, Schwartz Z, Differential regulation of growth plate chondrocytes by 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 involves cell maturation specific membrane receptor activated phospholipid metabolism. Crit Rev Oral Biol Med, in press
  4. Boyan BD, Sylvia VL, Dean DD, Schwartz Z 2001 24,25-(OH)2D3 regulates cartilage and bone via autocrine and endocrine mechanisms. Steroids 66: 363–374
  5. Pedrozo HA, Schwartz Z, Rimes S, Sylvia VL, Nemere I, Posner GH, Dean DD, Boyan BD 1999 Physiological importance of the 1,25-(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25-(OH)2D3. J Bone Miner Res 14:856–867[CrossRef][Medline]
  6. Nemere I, Schwartz Z, Pedrozo H, Sylvia VL, Dean DD, Boyan BD 1998 Identification of a membrane receptor for 1,25-dihydroxy vitamin D3 which mediates rapid activation of protein kinase C. J Bone Miner Res 13:1353–1359[CrossRef][Medline]
  7. Beno DWA, Brady LM, Bissonnette M, Davis BH 1995 Protein kinase C and mitogen-activated protein kinase are required for 1,25-dihydroxyvitamin D3-stimulated Egr induction. J Biol Chem 270:3642–3647[Abstract/Free Full Text]
  8. Binswanger U, Helmle-Kolb C, Forgo J, Mikic B, Murer H 1993 Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition. Pflugers Arch 424:390–397
  9. Baran DT, Sorenson AM, Shalhoub V, Owen T, Oberdorf A, Stein G, Lian JB 1991 1{alpha},25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium in clonal rat osteosarcoma cells lacking the vitamin D receptor. J Bone Miner Res 6:1269–1275[Medline]
  10. Baran DT, Quail JM, Ray R, Leszyk J, Honeyman T 2000 Annexin II is the membrane receptor that mediates the rapid actions of 1{alpha},25-dihydroxyvitamin D3. J Cell Biochem 78:34–46[CrossRef][Medline]
  11. Boyan BD, Bonewald LF, Sylvia VL, Nemere I, Larsson D, Norman AW, Rosser J, Dean DD, Schwartz Z 2002 Evidence for distinct membrane receptors for 1{alpha},25-(OH)2D3 and 24R,25-(OH)2D3 in osteoblasts. Steroids 67:235–246[CrossRef][Medline]
  12. Norman AW 1997 Rapid biological responses mediated by 1{alpha},25-dihydroxyvitamin D3: a case study of transcaltachia (the rapid hormonal stimulation of intestinal calcium transport). In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. San Diego: Academic Press; 233–256
  13. Nemere I, Ray R, McManus W 2000 Immunochemical studies on the putative plasmalemmal receptor for 1,25-(OH)2D3. I. Chick intestine. Am J Physiol 278:E1104–E1114
  14. Boyan BD, Posner GH, Greising DM, White MC, Sylvia VL, Dean DD, Schwartz Z 1997 Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway. J Cell Biochem 66:457–470[CrossRef][Medline]
  15. Song X, Bishop JE, Okamura WH, Norman AW 1998 Stimulation of phosphorylation of mitogen-activated protein kinase by 1{alpha},25-dihydroxyvitamin D3 in promyelocytic NB4 leukemia cells: a structure-function study. Endocrinology 139:457–465[Abstract/Free Full Text]
  16. Corvol M, Ulmann A, Garabedian M 1980 Specific nuclear uptake of 24,25-dihydroxycholecalciferol, a vitamin D3 metabolite biologically active in cartilage. FEBS Lett 116:273–276[CrossRef][Medline]
  17. Nemere I, Farach-Carson MC 1998 Membrane receptors for steroid hormones: a case for specific cell surface binding sites for vitamin D metabolites and estrogens. Biochem Biophys Res Commun 248:443–449[CrossRef][Medline]
  18. Sylvia VL, Schwartz Z, Schuman L, Morgan RT, Mackey S, Gomez R, Boyan BD 1993 Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures. J Cell Physiol 157:271–278[CrossRef][Medline]
  19. Capiati DA, Limbozzi F, Tellez-Inon MT, Boland RL 1999 Evidence on the participation of protein kinase C{alpha} in the proliferation of cultured myoblasts. J Cell Biochem 74:292–300[CrossRef][Medline]
  20. Morelli S, de Boland AR, Boland R 1993 Generation of inositol phosphates, diacylglycerol and calcium fluxes in myoblasts treated with 1,25-dihydroxyvitamin D3. Biochem J 289:675–679
  21. Vazquez G, de Boland AR 1996 Involvement of protein kinase C in the modulation of 1{alpha},25-dihydroxyvitamin D3-induced 45Ca2+ uptake in rat and chick cultured myoblasts. Biochim Biophys Acta 1310:157–162[Medline]
  22. Sylvia VL, Schwartz Z, Curry DB, Chang Z, Dean DD, Boyan BD 1998 1,25-(OH)2D3 regulates protein kinase C activity through two phospholipid-independent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes. J Bone Miner Res 13:559–569[CrossRef][Medline]
  23. de Boland AR, Morelli S, Boland R 1995 1,25-(OH)2-vitamin D3 stimulates phospholipase A2 activity via a guanine nucleotide-binding protein in chick myoblasts. Biochim Biophys Acta 1257:274–278[Medline]
  24. Schwartz Z, Boyan BD 1988 The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro. Endocrinology 122:2191–2198[Abstract]
  25. Swain LD, Schwartz Z, Boyan BD 1992 1,25-(OH)2D3 and 24,25-(OH)2D3 regulation of arachidonic acid turnover in chondrocyte cultures is cell maturation-specific and may involve direct effects on phospholipase A2. Biochim Biophys Acta 1136:45–51[Medline]
  26. Schwartz Z, Swain LD, Ramirez V, Boyan BD 1990 Regulation of arachidonic acid turnover by 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth zone and resting zone chondrocyte cultures. Biochim Biophys Acta 1027:278–286[Medline]
  27. Sylvia VL, Del Toro F, Dean DD, Hardin RR, Schwartz Z, Boyan BD 2001 Effects of 1{alpha},25-(OH)2D3 on rat growth zone chondrocytes are mediated by cyclooxygenase-1 and phospholipase A2. J Cell Biochem S36:32–45
  28. Schwartz Z, Swain LD, Kelly DW, Brooks BP, Boyan BD 1992 Regulation of prostaglandin E2 production by vitamin D metabolites in growth zone and resting zone chondrocyte cultures is dependent on cell maturation. Bone 13:395–401[Medline]
  29. Sylvia VL, Del Toro Jr F, Hardin RR, Dean DD, Boyan BD, Schwartz Z 2001 Characterization of PGE2 receptors (EP) and their role as mediators of 1{alpha},25-(OH)2D3 effects on growth zone chondrocytes. J Steroid Biochem Mol Biol 78:261–274[CrossRef][Medline]
  30. Vazquez G, Boland R, de Boland AR 1995 Modulation by 1,25-(OH)2 vitamin D 3 of the adenylyl cyclase/cyclic AMP pathway in rat and chick myoblasts. Biochim Biophys Acta 1269:91–97[Medline]
  31. Vazquez G, de Boland AR, Boland R 1997 1{alpha},25-(OH)2-vitamin D3 stimulates the adenylyl cyclase pathway in muscle cells by a GTP-dependent mechanism which presumably involves phosphorylation of G{alpha}i. Biochem Biophys Res Commun 234:125–128[CrossRef][Medline]
  32. Helm SH, Sylvia VL, Harmon T, Dean DD, Boyan BD, Schwartz Z 1996 24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms. J Cell Physiol 169:509–521[CrossRef][Medline]
  33. Del Toro Jr F, Sylvia VL, Schubkegel SR, Campos R, Dean DD, Boyan BD, Schwartz Z 2000 Characterization of PGE2 receptors (EP) and their role in 24,25-(OH)2D3-mediated effects on resting zone chondrocytes. J Cell Physiol 182:196–208[CrossRef][Medline]
  34. Cobb MH 1999 MAP kinase pathways. Prog Biophys Mol Biol 71:479–500[CrossRef][Medline]
  35. Morelli S, Buitrago C, Boland R, de Boland AR 2001 The stimulation of MAP kinase by 1,25-(OH)2-vitamin D3 in skeletal muscle cells is mediated by protein kinase C and calcium. Mol Cell Endocrinol 173:41–52[CrossRef][Medline]
  36. Whitmarsh AJ, Davis RJ 1996 Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74:589–607[CrossRef][Medline]
  37. Pang L, Sawada T, Decker SJ, Saltiel AR 1995 Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem 270:13585–13588[Abstract/Free Full Text]
  38. Schwartz Z, Lohmann CH, Vocke AK, Sylvia VL, Cochran DL, Dean DD, Boyan BD 2001 Osteoblast response to titanium surface roughness and 1{alpha},25-(OH)2D3 is mediated through the mitogen activated protein (MAP) kinase pathway. J Biomed Mater Res 56:417–426[CrossRef][Medline]
  39. Watanabe H, de Caestecker MP, Yamada Y 2001 Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-ß-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem 276:14466–14473[Abstract/Free Full Text]
  40. Kozawa O, Hatakeyama M, Kamiya Y, Kondo C, Matsuno H, Uematsu T 2001 Activation of p44/p42 mitogen-activated protein kinase limits triiodothyronine-stimulated alkaline phosphatase activity in osteoblasts. Biochem Biophys Res Commun 286:1140–1143[CrossRef][Medline]
  41. Norman AW, Okamura WH, Hammond MW, Bishop JE, Dormanen MC, Bouillon R, Van Baelen H, Ridal AL, Daane E, Khoury R, Farach-Carson MC 1997 Comparison of 6-s-cis and 6-s-trans locked analogs of 1{alpha},25-(OH)2-vitamin D3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis nor 6-s-trans locked analogs are preferred for genomic biological responses. Mol Endocrinol 11:1518–1531[Abstract/Free Full Text]
  42. Norman AW, Nemere I, Muralidharan KR, Okamura WH 1992 1ß,25 (OH)2-vitamin D3 is an antagonist of 1{alpha},25 (OH)2-vitamin D3 stimulated transcaltachia (the rapid hormonal stimulation of intestinal calcium transport). Biochem Biophys Res Commun 189:1450–1456[CrossRef][Medline]
  43. Nemere I, Ray R, Jia Z 1997 Further characterization of the basal-lateral membrane receptor for 1,25-(OH)2D3 in chick intestine. In: Norman AW, Bouillon R, Thomasset M, eds. Vitamin D: chemistry, biology and clinical applications of the steroid hormone. Riverside: University of California- Riverside Printing and Reprographic Services; 387–388
  44. Wheeler-Jones CP, May MJ, Houliston RA, Pearson JD 1996 Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E-selectin expression. FEBS Lett 388: 180–184
  45. Herbert JM, Augereau JM, Gleye J, Maffrand JP 1990 Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172:993–999[CrossRef][Medline]
  46. Kawamoto S, Hidaka H 1984 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochem Biophys Res Commun 125:258–264[CrossRef][Medline]
  47. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272