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D J Baylink

Publications and source records attributed to D J Baylink.

At least 145 records · Page 8Linked to original sources
PubMed1995

Human bone cell phenotypes differ depending on their skeletal site of origin.

This report describes skeletal site-related differences in human osteoblastic cell metabolism in studies of four patients. Northern analyses of the constitutive growth factor messenger ribonucleic acid (mRNA) expression pattern in mandibular and iliac crest-derived human osteoblastic cells (based on within-patient comparisons) revealed higher mRNA levels for strong mitogenic growth factors such as basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) in the rapidly proliferating and less alkaline phosphatase (ALP)-expressing mandibular osteoblastic cells compared to those in the lower bFGF and IGF-II mRNA levels in slowly proliferating iliac human osteoblastic cells exhibiting a higher ALP expression level. In contrast, transforming growth factor-beta (TGF beta) mRNA was more abundant in iliac human osteoblastic cells than in mandibular osteoblastic cells. Furthermore, we found that there was a proportionality, based on data from both sites, between the level of constitutive TGF beta mRNA and the response to exogenously administered bFGF or IGF-II. A comparable pattern of growth characteristics and mRNA expression was also observed in transformed human osteoblastic cells that had been subcloned in sublines expressing high and low levels of the human osteoblastic differentiation marker ALP. These findings are consistent with 1) skeletal site-related differences in human bone cell phenotypes, and 2) decreased IGF-II and bFGF expression and increased TGF beta expression and responsiveness to bFGF and IGF-II in human bone cells exhibiting a high ALP expression.

Alkaline Phosphatase↗
PubMed1995

Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids.

Insulin-like growth factor binding proteins (IGFBPs) serve as transport proteins for the IGFs and modulate their biological effects. In order to evaluate the relative contribution of IGFBP-5 to the IGF binding capacity of serum and other biological fluids and to study more accurately the in vivo regulation of serum IGFBP-5, we have developed an RIA for IGFBP-5 using recombinant human IGFBP-5 as the antigen, tracer, and standard. The validity of the IGFBP-5 RIA was established from the following data: 1) recombinant human IGFBP-5 and purified human bone-derived IGFBP-5 inhibited the binding of tracer to the antisera in an identical manner; 2) the immunoreactive IGFBP-5 in serum co-migrated with recombinant human IGFBP-5 in both sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance gel filtration chromatography; 3) none of the other known IGFBPs (IGFBP-1, -2, -3, -4, and -6) exhibited significant cross-reactivity; 4) the addition of IGF-I or IGF-II to the samples did not affect the recovery of IGFBP-5; and 5) the recovery of the exogenously added IGFBP-5 to serum was greater than 90%. In order to further validate our IGFBP-5 RIA, measurements of IGFBP-5 were carried out in the conditioned media samples collected from human bone cells treated with effectors that are known to influence IGFBP-5 production. Treatment of human bone cells with bone morphogenetic protein-7, which increases IGFBP-5 production, caused an increase in IGFBP-5 protein levels; dexamethasone, which decreases IGFBP-5 production, caused a decrease in IGFBP-5 protein levels measured in the conditioned medium by this RIA. Application of this RIA to the measurement of the IGFBP-5 level in human serum revealed that the circulating level of IGFBP-5 in healthy women decreased from 664 +/- 108 ug/L in prepubertal girls to 222 +/- 42 ug/L in women ages 61-85 yr (P < 0.001). There was also a small but significant decrease in the mean serum level of IGFBP-5 in women ages 23-39 yr compared with the level in prepubertal girls (589 +/- 66 vs. 664 +/- 108 ug/L, P < 0.05). Additional studies with this assay may provide an insight into the physiological mechanism that regulates IGFBP-5 production in vivo and the potential role of this protein as an IGF transport protein in serum and other biological fluids.

Adolescent↗
PubMed1995

Sequence comparison and predicted structure for the four exon-encoded regions of human insulin-like growth factor binding protein 4.

The IGFBPs bind to and modulate the function of the IGFs in various ways. Human IGFBP-4 inhibits IGF mediated cell proliferation. The IGFBP exon-encoded regions were aligned and secondary structure predictions for hIGFBP-4 were developed yielding predicted 3D co-ordinates for each such region of hIGFBP-4. The exon 1 encoded region is the most conserved among the IGFBPs. That of hIGFBP-4 is predicted as an array of beta-strands that include the glycine and cysteine rich IGFBP consensus pattern and that terminate with a helix. The exon 2 encoded region is the most variable among the IGFBPs. That of hIGFBP-4 is predicted as mostly an amphipathic helix. The remaining regions are also conserved among the IGFBPs. Those of hIGFBP-4 are also predicted to contain helices. The predicted structure of hIGFBP-4 comprises amino terminal beta-strands with four helices in the carboxy terminal two thirds of the molecule.

Amino Acid Sequence↗
PubMed1994

Evidence that human bone cells in culture contain binding sites for osteogenic protein-1.

Recent studies have shown that osteogenic protein (OP)-1 or bone morphogenetic protein (BMP)-7 increases proliferation and differentiation of human bone cells (HBCs) in culture and modulates production of IGF system components. In order to study the mechanism by which OP-1 causes these effects, we sought to test the hypothesis that the effects of OP-1 are mediated at least in part by specific receptors (for OP-1) in HBCs. Binding studies with serum-free cultures of normal HBCs and human osteosarcoma cells showed a maximum binding of 15-25% for [125I]OP-1; the binding was time- and temperature-dependent in different experiments. Scatchard analysis of [125]OP-1 binding to TE85 human osteosarcoma cells showed at least two binding sites, about 30,000 and 60,000 per cell with apparent Kd of 2.5 x 10(-10)M and 1 x 10(-9)M, respectively. [125]OP-1 binding to TE85 cells was displaced by unlabeled OP-1 (16-1000 ng/ml), with a 50% displacement at 250 ng/ml. BMP-2 effectively displaced [125]OP-1 binding to HBCs while TGF-beta 1 did not. Affinity cross-linking studies showed that [125]OP-1 interacted specifically with three binding sites with apparent M(r) of 34, 65 and > 205kDa. The findings of this study demonstrate that the effects of OP-1 on HBCs may be mediated in part via BMP-specific receptors.

Binding Sites↗
PubMed1994

Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption.

To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10(-6)-10(-12) M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Studies on the regulation of insulin-like growth factor binding protein 3 secretion in human osteosarcoma cells in vitro.

Previous studies demonstrated that insulin-like growth factors (IGFs) are important autocrine and paracrine mitogens for human bone cells in vitro and that IGF binding proteins (IGFBPs) are important regulators of the biologic actions of IGFs. Thus, the actions of IGFs may be determined not only by their concentrations but also by the type and amount of IGFBPs produced by human bone cells at a local site in bone. In this study, we sought to determine the effects of dexamethasone, 1,25-(OH)2D3, and parathyroid hormone (PTH) on the secretion of IGFBP-3 in human osteosarcoma cell lines. Serum-free cultures of low- and high-alkaline phosphatase (ALP) SaOS-2, MG-63, and TE89 human osteosarcoma cells were treated for 24 or 48 h with the effectors and the conditioned media used for determination of IGFBP-3 using a radioimmunoassay. We report that (1) the basal rate of IGFBP-3 secretion (ng/mg cellular protein) was dependent upon cell type, with TE89 > low-ALP Saos-2 > MG-63 > high-ALP SaOS-2 cells, and did not correlate with either basal cell proliferation or basal cellular ALP activity; (2) dexamethasone (10(-12)-10(-7) M) inhibited IGFBP-3 secretion in a dose-dependent manner in low-ALP SaOS-2, MG-63, and TE89 cells but not in high-ALP SaOS-2 cells; (3) 1,25-(OH)2D3 (10(-11)-10(-8) M) stimulated IGFBP-3 secretion in a dose-dependent manner in MG-63, low-ALP SaOS-2, and high-ALP SaOS-2 cells, and the coaddition of TGF-beta and 1,25-(OH)2D3 increased synergistically IGFBP-3 secretion and cellular ALP activity in MG-63 cells; and (4) human PTH-(1-34) (0.1-100 ng/ml) had no significant effect on IGFBP-3 secretion in MG-63, low-ALP SaOS-2, or high-ALP SaOS-2 cells. We conclude that such agents as dexamethasone, 1,25-(OH)2D3, and PTH differentially regulate IGFBP-3 secretion in human osteosarcoma cells in vitro.

Alkaline Phosphatase↗
PubMed1994

Development of a heterologous radioimmunoassay for canine osteocalcin.

The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (> 1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin cross-reacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The inter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

Combined magnetic fields increased net calcium flux in bone cells.

Low energy electromagnetic fields (EMF) exhibit a large number of biological effects. A major issue to be determined is "What is the lowest threshold of detection in which cells can respond to an EMF?" In these studies we demonstrate that a low-amplitude combined magnetic field (CMF) which induces a maximum potential gradient of 10(-5) V/m is capable of increasing net calcium flux in human osteoblast-like cells. The increase in net calcium flux was frequency dependent, with a peak in the 15.3-16.3 Hz range with an apparent bandwidth of approximately 1 Hz. A model that characterizes the thermal noise limit indicates that non-spherical cell shape, resonant type dynamics, and signal averaging may all play a role in the transduction of low-amplitude EMF effects in biological systems.

Alkaline Phosphatase↗
PubMed1994

The effects of the insulin-like growth factors and transforming growth factor beta on the Jun proto-oncogene family in MC3T3-E1 cells.

Previous studies have demonstrated that when cells of the mouse osteoblastic cell line MC3T3-E1 are exposed to IGF-I and IGF-II they exhibit rapid and transient induction of the transcript from the proto-oncogene c-fos [8]. To clarify the relationship between induction of cell proliferation and proto-oncogene expression in MC3T3-E1 cells, the acute affects of IGF-I and IGF-II, growth factors that stimulate cell proliferation, and of TGF-beta 1, which inhibits cell proliferation, northern analyses with cDNA-derived probes for the proto-oncogenes c-jun, jun-B, and jun-D were undertaken. Concurrent northern analyses with a probe for c-fos extended our previous results to include the effect of TGF-beta 1 on c-fos. IGF-I does not induce the c-jun, jun-B, or jun-D transcripts, the former and latter being produced at detectable levels constitutively. After 1 hour of exposure to IGF-II the c-jun transcript response ranges from onefold to 13-fold and the jun-D transcript response ranges around two-fold. After 1 hour of exposure to TGF-beta 1, the jun-B transcript response ranges from eightfold to 24-fold, the c-fos transcript response ranges between sixfold and sevenfold. The differences observed in the magnitude and kinetics of the induction provoked by these growth factors is consistent with the presence of a regulatory circuit acting through the Jun family members which may act to stimulate transcription differentially when bound to DNA either as homodimers or, with Fos family proteins, as heterodimers.

3T3 Cells↗
PubMed1994

Circulating levels of insulin-like growth factor-I and -II, and IGF-binding protein-3 in inflammation and after parathyroid hormone infusion.

In order to assess if the anabolic action of PTH is related to changes in circulating levels of insulin-like growth factor-I and -II (IGF-I and -II), and IGF binding protein 3 (IGFBP-3), 24 h of PTH infusion was performed in healthy women and in patients with rheumatoid arthritis (RA), a state where both bone metabolism and PTH secretion is influenced by the inflammatory activity. The patients with RA had lower basal levels of both IGF-I and -II than the healthy controls (P < 0.05). In neither group did the IGFs change after 24 h of PTH administration, while IGFBP-3 was significantly increased in the healthy controls (4600 +/- 1200 to 5750 +/- 2200 micrograms/l, P < 0.05). IGFBP-3 was not affected by PTH infusion in patients with RA when the disease had high activity, but when inflammation had subsided they responded with a similar increase in IGFBP-3 as the control group and basal IGF-I and -II levels were normalised. Since IGFBP-3 can enhance the anabolic action of IGF-I, increased IGFBP-3 levels after PTH infusion may reflect a mechanism by which PTH is anabolic for bone. Inflammation may inhibit bone formation via decreased serum levels of IGFs and blocked IGFBP-3 response to PTH.

Adult↗
PubMed1994

Evidence that human bone cells in culture produce insulin-like growth factor-binding protein-4 and -5 proteases.

Previous studies have shown that the actions of insulin-like growth factor-II (IGF-II) in bone are determined by both its concentration and the concentrations of the IGF-binding proteins (IGFBPs). As IGFBP concentrations may be regulated not only at the level of production, but also at the level of degradation, IGFBP proteases may be an important component of the IGF-II regulatory system. In this study, we have identified IGFBP-4 and IGFBP-5 protease activity in the conditioned medium (CM) of the human osteosarcoma U2 cell line (U2OS) and untransformed normal human bone cell (HBC) derived from skull. Proteolysis of the 29-kilodalton (kDa) [125I]IGFBP-5 produced an 18- to 20-kDa fragment of IGFBP-5, and 25 kDa [125I]IGFBP-4 yielded two lower mol wt fragments in the presence of IGF-II. CM from IGF-II-treated U2OS and normal HBC cultures exhibited decreased IGFBP-5 proteolytic activity compared to control cultures. In contrast, CM from IGF-II-treated HBC cultures had increased proteolytic activity against IGFBP-4. To determine the mechanisms by which IGF-II modulates IGFBP-4 and -5 proteolytic activity, CM from control U2OS cell culture was incubated with [125I]IGFBP-4 or -5 in the presence of various concentrations of IGF-II and IGF analogs under cell-free conditions. It was found that exogenous IGF-II stimulated IGFBP-4 proteolysis, but IGF analogs that had no or extremely low affinity to IGFBP-4 failed to induce IGFBP-4 proteolysis. On the contrary, exogenous IGF-II had no effect on IGFBP-5 proteolysis in cell-free U2OS CM. Both IGFBP-4 and IGFBP-5 proteolytic activities were inhibited by aprotinin, zinc chloride, and EDTA and eluted as a single major peak between mol wt markers of 160 and 67 kDa upon gel filtration. Based on the findings that HBCs in culture produce a protease(s) capable of cleaving both IGFBP-4 and IGFBP-5 and that IGF-II can promote or inhibit proteolytic degradation of IGFBP-4 and IGFBP-5, respectively, it is proposed that IGFBP protease(s) may be an important modulator of IGF activity in bone.

Bone and Bones↗
PubMed1994

Age-related decreases in insulin-like growth factor-I and transforming growth factor-beta in femoral cortical bone from both men and women: implications for bone loss with aging.

We determined the skeletal content of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF beta) in human bone as a function of age, using 66 samples of femoral cortical bone obtained from 46 men and 20 women between the ages of 20-64 yr. We found a linear decline in the skeletal content of IGF-I (nanograms per mg protein) with donor age (r = -0.43; P < 0.001) in the total population. The skeletal content of TGF beta also decreased with age (i.e. 1/TGF beta vs. age; r = 0.28; P < 0.02) for the total population. We did not observe any difference in the skeletal growth factor content between male and female donors. IGF-I content, when analyzed by decade divisions of age, showed a reduction between the 20- to 29-yr-old and the 50- to 59-yr-old subjects (P < 0.02). The loss rate of IGF-I was 1.56 ng/mg protein.yr, corresponding to a net loss of 60% of skeletal IGF-I between the ages of 20-60 yr. The loss rate of TGF beta was 0.03 ng/mg protein.yr, corresponding to a net loss of 25% of the skeletal TGF beta between the ages of 20-60 yr.

Adult↗
PubMed1994

[Autocrine regulation of cell proliferation and secretion of insulin-like growth factor I (IGF-I) in osteoblastic cell line MC3T3-E1].

Bone cells maintained in culture produce different growth factors which modulate cell growth via a mechanism of auto/paracrine regulation. IGF-1 is abundantly produced by murine bone cells where it acts as a mitogenic agent. The aim of this work was to study the effect of IGF-II, TGF beta 1, basic FGF (FGFb) and PDGF on cell growth and production of IGF-1 in the murine osteoblastic clonal cell line MC3T3-E1. IGF-1 was assayed by RIA after elimination of the IGF binding proteins. After 24th of treatment in culture conditions without serum, incorporation of [3H] methylthymidine increased significantly in MC3T3-E1 treated with IGF-II, FGFb and PDGF. The effect was dose-dependent. At low cell density (2.5 X 10(4) cemm/cm2) and after 24 h treatment, IGF-II at 10 ng/ml led to a 220% increase in IGF-I production in MC3T3-E1 cells (9.5 +/- 1.5 vs 4.2 +/- 0.44 ng/micrograms protein, p < 0.001) while TGF beta 1, FGFb and PDGF at 1 ng/ml led to a significant decrease (65, 95 and 85% respectively) in IGF-I (TGF beta 1: 1.5 +/- 0.3 ng/micrograms; FGBb: 0.21 +/- 0.04 ng/micrograms; PDGF: 0.66 +/- 0.1 ng/micrograms; p < 0.001). Production of IGF-I was controlled by a dose-dependent relationship and varied as a function of incubation time and cell density. IGF-II led to an increase in mRNA coding for IGF-1 as early as the first hour after IGF-II addition with a maximal effect at 6 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Growth factors and electromagnetic fields in bone.

Stimulation of bone by low-energy electromagnetic fields (EMFs) may prove to be an efficient therapy for skeletal disorders. The study discussed in this article demonstrates that an EMF of 15.3 Hz can increase the proliferation rate of an osteosarcoma cell line, TE-85. The increase in cell proliferation was associated with an increase in binding sites for insulin-like growth factor II and an increase in mitogen activity in culture media.

Bone Development↗
PubMed1994

Biochemical markers of bone turnover for the clinical assessment of bone metabolism.

Despite these potential problems, biochemical bone markers are the single most sensitive method for monitoring acute changes in bone metabolism. For example, subcutaneous injections of recombinant human insulin-like growth factor I cause a measurable increase in both procollagen and urinary DPD in as little as 1 day. Similarly, it is possible to measure a significant decrease in bone formation as determined by decreases in serum levels of ALP, OC, and C-PCP within 12 hours after the beginning of a PTH infusion study. Additionally, an increase in DPD/cr was determined within 24 hours of the start of bed rest. These changes, seen within 24 hours, are far earlier than could be detected by any other method of monitoring bone metabolism. Thus, biochemical assays have opened a new era where changes in bone metabolism can be detected in hours to days. This acute detectability should be especially helpful to the development of new drugs and the optimization of the use of approved drugs. Accordingly, definite dose-response studies can now be done in a reasonable time. For osteoporosis therapy there are reasons to consider cyclic drug administration, such as avoiding drug resistance (PTH or calcitonin), avoiding overtreatment (bisphosphonates), or avoiding a possible mineralization defect (fluoride). By using biochemical assays, we can determine the optimum amount of "on time" and "off time" in cyclic therapy. Of the bone formation assays, ALP, OC, and PCP, we recommend for routine use the OC assay because of its high discriminant power and because it has been better characterized, in terms of clinical application, than the PCP assays and the ALP IRMA. If, however, the serum cannot be drawn at a specific time in all patients to be studied, we recommend the ALP assay because, unlike the OC assay, it shows no diurnal variation. Of the bone resorption assays, HYP, TRAP, GHYL, and PYD/DPD, we recommend the urine PYD/DPD assay (adjusted for creatinine) because it is commercially available and because, along with the urine GHYL assay, it is the most sensitive bone resorption assay. Established guidelines for the use of assays in patient care is not yet available, largely because of the large intrapatient variation seen with most assays. Once this problem is resolved, it should be possible to apply biochemical assays to routine clinical practice. For example, if the patient has a urine DPD/cr (indicating a high bone resorption rate), the patient would be selected for antiresorptive therapy, and subsequently the urine DPD/cr assay would be repeated during therapy to determine the effective dose of the drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1994

Quantification of skeletal alkaline phosphatase in osteoporotic serum by wheat germ agglutinin precipitation, heat inactivation, and a two-site immunoradiometric assay.

Three methods for quantifying skeletal alkaline phosphatase (ALP; EC 3.1.3.1) activity/immunoactivity in serum--heat inactivation, wheat germ agglutinin (WGA) precipitation, and an immunoradiometric assay--were tested for recovery and specificity and applied to 81 sera collected from 14 postmenopausal osteoporotic subjects. The heat-inactivation and WGA precipitation assays showed relative recoveries of 91-100% and 16-32%, respectively, for skeletal ALP with complete specificity (no cross-reactivity with hepatic or intestinal ALP); the IRMA showed a relative recovery of 86-100% and 11-14% cross-reactivity with hepatic ALP. There was a closer correlation between the heat-inactivation assay and IRMA (r = 0.833) than between the WGA precipitation assay and IRMA (r = 0.673) or between the heat-inactivation and WGA-precipitation assays (r = 0.568). The WGA precipitation assay failed to detect skeletal ALP in three serum samples that contained significant amounts as determined by the heat-inactivation assay and the IRMA.

Alkaline Phosphatase↗
PubMed1993

Osteogenic protein-1 stimulates proliferation and differentiation of human bone cells in vitro.

The effect of recombinant human osteogenic protein-1 (OP-1) on proliferation and alkaline phosphatase (ALP) activity was studied using human bone cells in culture. TE85, osteosarcoma cells with osteoblastic characteristics and normal bone cells derived from mandible (HBM) were used. OP-1 stimulated 3H-thymidine incorporation in a dose dependent manner in TE85 (5-fold over vehicle control at 3-10 ng/ml) and HBM cells (1.8-fold at 100 ng/ml). In TE85 cells, OP-1 also increased cell number (2.4-fold over vehicle control at 3 ng/ml). OP-1 stimulated ALP activity in TE85 cells (4-fold over vehicle control at 30 ng/ml), but moderately inhibited ALP activity in HBM cells (to 67% of vehicle control at 100 ng/ml). Because 1,25(OH)2D3 has been shown to increase ALP activity in many cell types, we also studied if 1,25(OH)2D3 modulates the effects of OP-1 on ALP activity. In the presence of 10(-8) M 1,25(OH)2D3, a biphasic response occurred in TE85 and HBM cells with stimulation of ALP activity at low dose of OP-1 and inhibition at high dose. Thus, the effect of OP-1 on ALP activity appeared to be modulated by 1,25(OH)2D3. Our results suggest that OP-1 could be an important regulator of osteoblast proliferation and differentiation.

Alkaline Phosphatase↗