1alpha ,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells

Naibedya Chattopadhyay, R. J. MacLeod, Jacob Tfelt-Hansen, and Edward M. Brown

Endocrine-Hypertension Division and Membrane Biology Program, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1alpha ,25(OH)2-vitamin D3 [1,25(OH)2D3] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)2D3 inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)2D3 followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)2D3. Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)2D3 on MG-63 cells can be accounted for by the concomitant 1,25(OH)2D3-induced inhibition of HGF production.

osteoblast; proliferation; autocrine; mitogen-activated protein kinase; tyrosine kinase; hepatocyte growth factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BY BINDING TO THE VITAMIN D receptor (VDR), 1,25 (OH)2-vitamin D3 [1,25(OH)2D3] directly activates or represses gene expression through vitamin D-responsive elements (VDRE) located in the promoter regions of various target genes (31). The VDR can function as a homodimer (VDR-VDR), but it more often heterodimerizes with the retinoid-X-receptor (RxR) and modulates cellular proliferation and differentiation of osteoblastic cells (17). The MG-63 cell line is a human osteoblast-derived osteosarcoma cell line and is considered to be representative of a particular subpopulation of osteoblasts, i.e., osteoblast precursors or early undifferentiated osteoblast-like cells (11). Although MG-63 cells are not capable of forming mineralized bone, they differentiate after treatment with 1,25(OH)2D3 or transforming growth factor-beta to the point of matrix maturation after the ordered expression of bone matrix proteins (e.g., sequentially expressing collagen type I, alkaline phosphatase, and osteocalcin). Induction of differentiation by 1,25(OH)2D3 takes place via cell cycle arrest at the G1 phase through p53-independent induction of Waf1/Cip1, which inhibits cyclin-dependent kinase (42).

Hepatocyte growth factor (HGF) plays an essential role in the development and regeneration of the liver and also stimulates the growth, motility, and morphogenesis of a variety of cell types (22). Although initially thought to be of mesodermal origin, HGF is expressed almost ubiquitously, including by osteoblasts (7,37), osteoclasts (15), and chondrocytes (16). It exerts autocrine and/or paracrine effects depending on the distribution of its receptor, the protooncogene c-Met, which is a tyrosine kinase-activated receptor (29). This receptor is expressed by numerous cells, among which are normal human osteoblasts and osteoclasts (8, 15) as well as various tumors of skeletal origin (12, 14), indicating that HGF may regulate bone metabolism under normal and pathological conditions. HGF is mitogenic for cells of both the osteoblast and osteoclast lineages, thereby potentially providing a mechanism for coordinating bone resorption and formation through an autocrine-paracrine loop(s) (7, 15). In addition, it has been postulated to participate in bone remodeling and repair (15).

Similar to its expression by several other malignant tumors, c-Met is also overexpressed by various musculoskeletal tumors, including osteosarcomas (3, 35). These tumors are likewise known to express and secrete HGF, thereby suggesting the existence of an autocrine mode of action for HGF. The resultant stimulation of proliferation, migration, inhibition of apoptosis, and angiogenesis by HGF could all give rise to further tumor progression. However, in the case of osteosarcomas, the evidence for an autocrine mode of action for HGF is circumstantial, consisting of only evidence of expression of HGF and c-MET in these tumors.

In this study, using the MG-63 osteoblast-like cell line, we unequivocally demonstrate that HGF functions in an autocrine manner, resulting in mitogenesis. Increased mitogenesis is achieved by an active process whereby c-Met and subsequently the mitogen-activated protein kinase (MAPK) pathway are constitutively activated. Furthermore, we show that the inhibitory effect of 1,25(OH)2D3 on the proliferation of MG-63 cells (13, 32) is likely explained by the concomitant 1,25(OH)2D3-induced inhibition of HGF production.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. All routine culture media were obtained from GIBCO-BRL (Grand Island, NY), and FBS was from Hyclone (Logan, UT). The MG-63 cells line, established as an osteoblastic cell line from a human osteosarcoma, was obtained from Dr. Nancy Weigel, Baylor College of Medicine (Houston, TX), who obtained this cell line originally from the American Type Culture Collection (Manassas, VA). MG-63 cells were grown in alpha -MEM (1.8 mM Ca2+, 0.81 mM Mg2+, and 1.0 mM H2PO4) supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO2 at 37°C.

1,25(OH)2D3 in ethanol was purchased from Biomol (Plymouth Meeting, PA); antibodies against phosphorylated and nonphosphorylated mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2 were from Cellular Signaling (Beverly, MA); c-Met antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-phospho-Tyr antibody (PY 20) was from Transduction Laboratories (Lexington, KY). Anti-human HGF polyclonal antibody and the HGF ELISA kit were purchased from R&D Systems (Minneapolis, MN). Rabbit IgG was from Sigma Chemical (St. Louis, MO).

Determination of HGF secretion. For studying HGF secretion, MG-63 cells were grown to 70-75% confluence in complete growth medium in 24-well plates. They were then serum starved overnight in growth medium minus FBS containing 0.2% BSA along with various concentrations of 1,25(OH)2D3 (10-9 to 10-6 M). Medium samples were cleared by centrifugation, and HGF was measured in this conditioned medium with an ELISA. The ELISA employs a quantitative sandwich, enzyme-linked immunoassay technique, utilizing a monoclonal antibody specific for HGF that is bound to microtiter wells. Assay sensitivity was 125 pg/ml. Data are expressed as picograms per microgram protein.

Northern blot analysis. To study whether 1,25(OH)2D3 exerted an inhibitory effect on the expression of HGF mRNA, we performed Northern blot analysis as described previously (9). Briefly, cellular RNA was isolated (10) using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The RNA recovered was quantitated by spectrophotometry, and aliquots of 25 µg total RNA from control or test samples were loaded on a formaldehyde agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA to document equal loading of RNA from the various experimental samples. The RNA was then blotted on nylon membranes (Duralon; Stratagene, La Jolla, CA). A cDNA probe for HGF was kindly provided by Dr. T. Nakamura (Osaka University School of Medicine, Osaka, Japan). A 2.3-kbp fragment of human HGF was subcloned into pBlueScript SK(-) and was digested with BamHI and KpnI to excise a 2.2-kbp fragment, which was then used for probing the Northern blots with a standard technique (9). Specific radioactive signals were analyzed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) with the ImageQuant program.

Semiquantitative RT-PCR of c-Met mRNA. The same RNA samples utilized for Northern analysis of HGF as described above were also used for semiquantitative determination of c-Met mRNA. For semiquantitative RT-PCR, the one-step RT-PCR kit was obtained from Qiagen, and experiments were performed using the manufacturer's protocol. Briefly, 2.0 µg of total RNA were mixed with a cocktail of RT and PCR reaction buffers, including 400 µM of each dNTP, 1.5 mM MgCl2, and 0.6 µM of the gene-specific primers pairs [c-Met: 5'-GCCTCTGGTTCCCCTTCAATAG-3' (sense), 5'-TTATCATCAAAGCCCTTGTCGG-3' (antisense); beta -actin: 5'-TTGTAACCAACTGGGACGATATGG-3' (sense), 5'-GCTGGGGTGTTGAAGGTCTCAAAC-3' (antisense)] and a one-step RT-PCR enzyme mix (Omniscript and Sensiscript RTs and HotStart Taq DNA polymerase).

The thermal cycler conditions were as follows: 1) RT at 50°C for 30 min, 2) initial PCR activation step at 95°C for 15 min, and 3) three-step cycling (total number of cycles 25), denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min. Aliquots (10 µl) of the reaction were then run in 1.5% agarose gels for visualization of the products.

Proliferation assay. MG-63 cells were trypsinized from a 75-cm2 flask maintained in growth medium and then seeded in 24-well plates at a density of 105/well in a volume of 0.5 ml growth medium. They were cultured for 24 h and then serum starved for 24 h, after which HGF (1-5 ng/ml) was added along with 25 µl [3H]thymidine (50 µCi/ml), and the cells were cultured again for 24 h. [3H]thymidine incorporation was measured by removing the medium and lysing the cells with 0.5 ml of 10% trichloroacetic acid. The resultant DNA pellet was dissolved in 0.5 ml of 200 mM NaOH. Each point shown in RESULTS represents data obtained from six wells in one out of three similar, independent experiments.

To study the effects of neutralizing antibody against HGF on cellular proliferation, cells were seeded in 24-well plates, as described above. Dilutions (1:100 and 1:50) of 0.1 mg/ml rabbit anti-HGF antibody or similar concentrations of rabbit IgG were added to serum-free medium and then added to the cells. Medium was removed after 12 h, and anti-HGF antibody or IgG was added again for a second time. On a third occasion, 12 h after the second addition, anti-HGF antibody or IgG was added again to the cells along with 25 µl [3H]thymidine (50 µCi/ml), and the cells were cultured for an additional 12 h. [3H]thymidine incorporation into MG-63 cells treated with anti-HGF antibody or rabbit IgG was subsequently determined by the same method described earlier. Each point shown in RESULTS represents data obtained from six wells in one out of three similar, independent experiments.

To further study the mitogenic action of HGF, we treated two groups of 50-60% confluent MG-63 cells with 10-7 M 1,25(OH)2D3 for 72 h. We then continued the 1,25(OH)2D3 treatment in one group of cells by fresh addition of 1,25(OH)2D3 while adding 5 ng/ml HGF to the other in the continued presence of 1,25(OH)2D3 for 48 h, at which stage 25 µl [3H]thymidine (50 µCi/ml) were added to both groups and to the vehicle control group. Cells were lysed and assayed for [3H]thymidine incorporation following the procedure described above. Each point shown in RESULTS represents data obtained from six wells in one out of three similar, independent experiments.

Western blotting and immunoprecipitation. For the determination of MEK1 or Erk1/2 phosphorylation, monolayers of MG-63 cells were grown on six-well dishes. Cells were cultured in growth medium until 70% confluence was reached. They were then incubated with anti-HGF neutralizing antibody (1:50 dilution of a 0.1 mg/ml stock) or with an equal amount of rabbit IgG, which was each replaced three times at 12-h intervals as described above in the proliferation assay. At the end of the third incubation with antibody or IgG, the medium was removed, and the cells were washed two times with PBS and then incubated with HGF (5 ng/ml) for the times indicated in RESULTS for Western blotting and immunoprecipitation. After this incubation, the cells were washed two times with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF. Next, 100 µl ice-cold lysis buffer were added [20 mM Tris · HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerophosphate, and a cocktail of protease inhibitors]. The protease inhibitors were as follows: 10 µg/ml each of aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor as well as 100 µg/ml Pefabloc, which were added from frozen stocks. The sodium vanadate, NaF, and Pefabloc were prepared fresh on the day of the experiment. The cells were scraped in the lysis buffer, sonicated for 5 s, and centrifuged at 10,000 g for 5 min at 4°C, and the supernatants were frozen at -20°C. After being thawed, equal amounts of supernatant protein (100 µg) were separated by SDS-PAGE. Each time, protein samples were run in two parallel gels (one for determination of phosphorylated proteins and the other for the nonphosphorylated proteins). The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell) and stained with Ponceau red for determination of equal loading. Blots were then incubated with blocking solution (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.25% BSA) containing 5% dry milk for at least 1 h at room temperature. Phospho-p44/42 MAPK and phospho-MEK1 antibodies as well as antibodies to the nonphosphorylated proteins (1:1,000 dilution for all) were detected by immunoblotting using polyclonal antibodies against the respective antigens after an 18-h incubation at 4°C. Blots were washed five times for 15 min each at room temperature (1% PBS, 1% Triton X-100, and 1% dry milk) and then incubated for 1 h with a second, goat anti-rabbit, peroxidase-linked antiserum (1:1,000) in blocking solution. Blots were washed again (5 times for 15 min each), and bands were visualized by chemiluminescence according to the manufacturer's protocol (Supersignal; Pierce Chemical). Quantitative comparisons of the phosphorylation of MEK1 or Erk1/2 were made using an ImageQuant and a Personal Densitometer (Molecular Dynamics). Protein concentrations were measured using the micro-bicinchoninic acid protein kit (Pierce).

For immunoprecipitation of c-Met, MG-63 cells were treated for 48 h with neutralizing HGF antibody as done for the studies of MAPK activation and were then incubated with HGF (10 ng/ml) in the serum-free medium described under Proliferation assay containing 0.2% BSA, 4 mM L-glutamine, and 0.5 mM CaCl2. Preparation of cell lysates was performed following the same protocol as for Western analysis. Lysates were centrifuged at 14,000 revolutions/min (rpm) for 10 min, and the pellet was discarded. Samples were assayed for protein content as above, and 0.5 mg of protein was added to 1 µg of c-Met primary antibody and incubated for 18 h at 4°C followed by a 90-min incubation with protein A-agarose. Lysates were then briefly centrifuged, and the supernatant was discarded. Pellets were washed three times in lysis buffer and one time in PBS, resuspended in 45 µl of 2× gel-loading buffer, boiled for 10 min, and centrifuged for 5 min at 14,000 rpm, and then the supernatant was applied to a 10% SDS-polyacrylamide gel. Tyrosine phosphorylation of c-Met was detected using an 18-h incubation with a 1:1,000 dilution of a rabbit anti-phosphotyrosine antibody (Transduction Laboratories). After being washed, the membranes were incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; Sigma), and immunoreactive proteins were detected as described above.

Statistics. Results are expressed as means ± SE. Statistical evaluation for differences between group means was carried using one-way ANOVA followed by Fisher's protected least significant difference. For all statistical tests, values of P < 0.05 were considered to indicate a statistically significant difference between groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1,25(OH)2D3 inhibits HGF synthesis. Before determination of secreted HGF in the conditioned medium of MG-63 cells after treatment with 1,25(OH)2D3, cells were incubated for 18 h in appropriate serum-deprived medium containing 0.2% BSA as described in MATERIALS AND METHODS. Cells were then incubated with vehicle (absolute ethanol) or increasing concentrations of 1,25(OH)2D3 from 10-9 to 10-6 M for another 18 h. Treatment with 10-9 M 1,25(OH)2D3 did not show any effect on HGF secretion in conditioned medium (data not shown). However, treatment with 10-8 to 10-6 M 1,25(OH)2D3 inhibited HGF secretion markedly (Fig. 1A).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   A: dose-dependent inhibition of hepatocyte growth factor (HGF) secretion from MG-63 cells by 1alpha ,25(OH2)-vitamin D3 [1,25(OH)2D3]. MG-63 cells were incubated with 10-8 to 10-6 M 1,25(OH)2D3 for 18 h, and conditioned media were collected and subjected to HGF determination by ELISA, as described in MATERIALS AND METHODS. V, vehicle. a, Significantly lower than V (P < 0.05); b, significantly lower than V and 10-8 M treated 1,25(OH)2D3 (P < 0.05). B: Northern analysis of HGF mRNA after treatment similar to that described in A. Total RNA (25 µg) was probed with HGF cDNA (32P labeled). Lane 1, vehicle; lane 2, 10-8 M 1,25(OH)2D3; lane 3, 10-7 M 1,25(OH)2D3; lane 4, 10-6 M 1,25(OH)2D3. Arrow indicates 6.2-kb HGF mRNA. C: ethidium bromide-stained gel of the RNA samples showing equal loading. D: semiquantitative determination of c-Met mRNA in cells treated with 1,25(OH)2D3 by RT-PCR using the beta -actin housekeeping gene as a control, as described in MATERIALS AND METHODS. Lane 1, vehicle; lane 2, 10-7 M 1,25(OH)2D3; lane 3, 10-6 M 1,25(OH)2D3. L, molecular weight marker.

To determine whether 1,25(OH)2D3 exerted an inhibitory effect on the synthesis of HGF mRNA, RNA samples were extracted from cells treated as above with 1,25(OH)2D3, and the Northern blots were probed with a human HGF cDNA. Figure 1B shows that 10-6 M 1,25(OH)2D3 maximally reduced the level of a single 6.2-kb HGF transcript. Inhibition of HGF gene expression was dose dependent and paralleled the pattern obtained in the secretion experiments. Figure 1C shows a photograph of an ethidium bromide-stained gel of the total RNA samples run for the Northern analysis shown in Fig. 1B, demonstrating equal loading of the samples.

We then studied whether or not 1,25(OH)2D3 treatment alters c-Met expression, since HGF is known to act in an autocrine manner in various cells. For this purpose, we used semiquantitative PCR with previously published primer pairs for the HGF transcript and the human beta -actin gene. The treatment of MG-63 cells was similar to that described earlier for studying the secretion and mRNA levels of HGF. Figure 1D shows that there were no changes in the intensities of the PCR-amplified bands corresponding to c-Met transcript from the reverse-transcribed RNA samples obtained from MG-63 cells treated with 10-7 and 10-6 M 1,25(OH)2D3. Normalization of the mRNA quantity in each sample was carried out by performing gene-specific RT-PCR (i.e., c-Met and beta -actin mRNAs) simultaneously in each reaction.

Expression of functional HGF receptor, c-Met, in MG-63 cells. To confirm that the expressed c-Met mRNA (Fig. 1D) was translated into a functional receptor, we studied ligand-induced phosphorylation of tyrosine in c-Met. Because HGF is secreted robustly by MG-63 cells, which also express the receptor for HGF, c-Met, we anticipated the existence of an autocrine mode of action of HGF in these cells, thereby resulting in constitutive activation of c-Met. Our pilot studies revealed that c-Met was phosphorylated on tyrosine residues even without the addition of HGF. Therefore, we neutralized endogenously secreted HGF for 48 h with neutralizing antibody (added fresh every 12 h) or added an equal amount of rabbit IgG as a negative control and then added HGF as described in MATERIALS AND METHODS. Imunoprecipitation of c-Met with a polyclonal anti-c-Met antibody followed by subsequent immunoblotting and probing with a monoclonal anti-phospho-tyrosine antibody revealed phosphorylation of c-Met (Fig. 2).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   A and B, top, show results of immunoprecipitation of c-Met followed by probing with an anti-phosphotyrosine antibody (PY 20). A: lane 1, c-Met is constitutively tyrosine phosphorylated in MG-63 cells treated with anti-rabbit IgG; lane 2, neutralizing HGF using polyclonal neutralizing antibody abolishes phosphorylation; lane 3, treatment of cells with HGF after neutralization of endogenous HGF promotes tyrosine phosphorylation of c-Met. B: time course of c-Met phosphorylation by HGF after neutralization of endogenous HGF. Cells were treated with neutralizing antibody (1:50 dilution of 0.1 mg/ml stock; lane 1) and addition of 5 ng/ml HGF for 2 min (lane 2), 5 min (lane 3), and 10 min (lane 4). c-Met activation is rapid, since phosphorylation is significantly reduced at 5 and 10 min compared with 2 min. A and B, bottom, show comparable expression of c-Met protein in all lanes.

Figure 2A, lane 1, top, shows phosphotyrosine immunoreactivity in immunoprecipitated c-Met from MG-63 cells treated with rabbit IgG, which was completely abolished in cells treated with anti-HGF neutralizing antibody (lane 2). Lane 3 shows tyrosine phosphorylation of c-Met in MG-63 cells treated for 48 h with the neutralizing antibody as in lane 2, followed by a 2-min treatment with HGF (5 ng/ml). Nonphosphorylated c-Met in Fig. 2A, bottom, documents that there was equal addition of immunoprecipitated c-Met in each lane.

The time course of c-Met phosphorylation was rapid, as revealed in Fig. 2B. There was nearly undetectable phosphotyrosine immunoreactivity in lane 1, top, which was derived from cells treated with neutralizing HGF antibody followed by c-Met immunoprecipitation. In contrast, there was a robust phosphotyrosine band in lane 2, top, after treatment of cells with HGF for 2 min that had been previously treated with neutralizing antibody to HGF. Longer incubation (5 and 10 min) of the cells with HGF (lanes 3 and 4) showed significant attenuation of the degree of phosphorylation of c-Met, although the extent of phosphorylation was clearly greater than that observed in lane 1. Probing of the blot with an antibody to nonphosphorylated c-Met (Fig. 2, bottom) revealed equal loading of immunoprecipitated c-Met in each lane.

Neutralizing HGF inhibits cellular proliferation. Confluent cells (60-70%) were first incubated overnight in serum-deprived medium containing 0.2% BSA. This medium was then replaced with fresh medium containing 1-5 ng/ml HGF plus [3H]thymidine for 24 h. In agreement with our hypothesis that HGF acts in an autocrine fashion, we observed no change in cellular proliferation in MG-63 cells in response to HGF compared with control cells (Fig. 3A). However, after neutralization of endogenous HGF with a specific rabbit, anti-HGF antibody (28, 38), we observed inhibition of cellular proliferation of MG-63 cells compared with the control cells treated with a similar concentration of rabbit polyclonal IgG, as assessed by [3H]thymidine incorporation into DNA (Fig. 3B).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   A: addition of HGF does not cause proliferation of MG-63 cells. B: neutralization of endogenous HGF following the method described in MATERIALS AND METHODS inhibits proliferation of MG-63 cells compared with IgG-treated control cells. Data obtained from 2 independent experiments where each treatment point consisted of 6 wells. * Significantly lower than IgG-treated cells (P < 0.05) and ** significantly lower than IgG and 1:100 dilution of antibody-treated cells (P < 0.05).

HGF contributes to 1,25(OH)2D3-induced inhibition of cellular growth. We observed that endogenously secreted HGF contributes to the proliferation of MG-63 cells as noted above and that neutralizing this cytokine with a specific antibody inhibits their proliferation. Because 1,25(OH)2D3 inhibits the secretion of HGF to an essentially undetectable level at a concentration of 10-7 M over a period of 2-3 days, we used this approach to remove endogenous HGF. After a 72-h incubation with 1,25(OH)2D3 (10-7 M), we incubated one group of cells with both 1,25(OH)2D3 and 5 ng/ml HGF for 48 h and compared the proliferation of these cells with those treated with vehicle or with 1,25(OH)2D3 alone. Figure 4 shows that 1,25(OH)2D3 treatment results in ~50% inhibition of proliferation, whereas addition of HGF reverses the inhibitory effect of 1,25(OH)2D3 on the proliferation of these cells.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   1,25(OH)2D3 induces growth inhibition of MG-63 cells by reducing HGF production. Confluent cells (50-60%) were treated with 10-7 M 1,25(OH)2D3 or vehicle for 72 h. 1,25(OH)2D3 and 1,25(OH)2D3 plus HGF (5 ng/ml) were then added to two groups of cells while one group was continued as a vehicle control. At this point, [3H]thymidine was added, and the cells were cultured for 48 h. Cells were then lysed, and incorporation of [3H]thymidine was measured as described in MATERIALS AND METHODS. Although 1,25(OH)2D3 treatment inhibits proliferation of these cells (hatched bar) to 50-55% compared with vehicle control (open bar), HGF restores proliferation to the control level (filled bar). Data obtained from 2 independent experiments where each treatment point consisted of 6 wells. * Significantly less than with 2 treatments (P < 0.05).

HGF-induced cellular mitogenesis involves the MAPK pathway. We then studied the possibility of MAPK activation in the HGF-induced mitogenesis of MG-63 cells. As described above, the high endogenous level of HGF maintains its receptor, c-Met, in a chronically activated state. Presumably because of this activation, we observed constitutive phosphorylation of Erk1/2 [phospho-p44/42 MAPK (Thr202/Tyr204); Fig. 5A]. However, addition of HGF after neutralization of endogenous HGF resulted in rapid phosphorylation of Erk1/2 (Fig. 5B). We further observed that MEK1/2, the kinase immediately upstream of Erk1/2, was also phosphorylated by HGF under the conditions used for studying the phosphorylation of Erk1/2.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5.   A: extracellular signal-regulated kinase (Erk) 1/2 [p44/42 mitogen-activated protein kinase (MAPK)] is constitutively phosphorylated in MG-63 cells treated with anti-rabbit IgG, as observed in lane 1, whereas treatment of cells with the polyclonal anti-HGF neutralizing antibody abolishes phosphorylation (lane 2). Incubation of MG-63 cells pretreated with neutralizing antibody with 5 ng/ml HGF rapidly induces phosphorylation of Erk1/2 (lanes 3-6). Lane 3, cells treated with HGF neutralizing antibody as described in MATERIALS AND METHODS. HGF treatment for 5 min (lane 4), 10 min (lane 5), and 15 min (lane 6). Equivalent levels of nonphosphorylated Erk2 in all lanes indicating equal protein loading are shown on bottom. B: incubation of HGF-neutralized MG-63 cells with 5 ng/ml HGF rapidly induces phosphorylation (p) of mitogen/extracellular signal-regulated kinase (MEK) 1, the kinase upstream of Erk1/2. Lane 1, cells treated with HGF neutralizing antibody as described in MATERIALS AND METHODS. HGF treatment for 5 min (lane 2), 10 min (lane 3), or 15 min (lane 4). Equivalent levels of nonphosphorylated MEK 1 in all lanes indicating equal loading of each protein sample are on bottom. C: downregulation of HGF secretion resulting from 1,25(OH)2D3 treatment results in inhibition of basal phosphorylation of Erk1/2. Cells were treated with varying concentrations of 1,25(OH)2D3 for 24 h (lanes 1-3) or 48 h (lanes 4-6). Lane 1, vehicle; lane 2, 10-7 M 1,25(OH)2D3; lane 3, 10-6 M 1,25(OH)2D3; lane 4, vehicle; lane 5, 10-7 M 1,25(OH)2D3; lane 6, 10-6 M 1,25(OH)2D3. Equivalent levels of nonphosphorylated Erk2 in all lanes indicating equal loading of each protein sample are on bottom.

As an alternative approach to show that HGF signaling results in activation of p44/42 MAPK, we incubated MG-63 cells with 10-7 and 10-6 M 1,25(OH)2D3 for 24 or 48 h, at which time endogenous HGF secretion is downregulated to nearly undetectable levels, and then determined the level of phospho-Erk1/2 (Fig. 5C). Our data show a dose- and time-dependent inhibition of the constitutive phosphorylation of Erk1/2 by 1,25(OH)2D3, which did not involve any change in protein synthesis since the level of nonphosphorylated Erk (Erk2) was comparable from sample to sample.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HGF modulates bone cell proliferation and function and serves as a coupling factor in bone remodeling. Simultaneous expressions of HGF and its receptor c-Met have been demonstrated in various bone tumors, including osteosarcomas (35, 14). Although these observations, similar to tumors derived from a variety of other tissues, strongly implicate HGF in the progression of malignancies arising from bone, nevertheless, a direct autocrine action of HGF has not been demonstrated in bone cells. In this report, we show that MG-63 human osteosarcoma cells secrete copious amounts of HGF. Although addition of exogenous HGF does not alter the proliferation of MG-63 cells under basal conditions, neutralization of endogenously secreted HGF with a neutralizing antibody substantially inhibits proliferation, as would have been expected after interruption of the autocrine loop. In addition, inhibition of HGF synthesis/secretion by 1,25(OH)2D3 treatment followed by addition of HGF reverses the cellular growth inhibition caused by the 1,25(OH)2D3 to the control level. This shows that one of the mechanisms underlying the inhibition of proliferation of MG-63 by 1,25(OH)2D3 is via inhibition of the synthesis of the proproliferative cytokine HGF.

Therefore, we hypothesized an autocrine mode of HGF-induced cellular proliferation in MG-63 cells. This possibility was reinforced further by the detection of c-Met transcript in these cells, as assessed by RT-PCR. To investigate the functionality of this expressed HGF receptor, we tested ligand (HGF)-mediated phosphorylation of c-Met, a receptor tyrosine kinase. First, we observed that c-Met is constitutively phosphorylated, most likely because of a high endogenous secretion of HGF, although we cannot rule out transactivation of c-Met by some unknown membrane receptor, including putative G protein-coupled receptors. Our hypothesis that HGF is solely responsible for constitutive c-Met expression was confirmed, since blocking the availability of endogenously secreted HGF with a specific neutralizing antibody abolished phosphorylation of c-Met. Furthermore, freeing c-Met sites previously occupied with endogenous HGF through neutralization of HGF resulted in rapid tyrosine phosphorylation of c-Met after addition of exogenous HGF. The rapidity of this time course resembles that observed in H441 human alveolar cells (4).

Having confirmed the role of HGF in mitogenesis, we sought to study whether or not MAPK such as MEK1/2 are activated by HGF. As observed previously in the case of various malignant tumors, such as gastric adenocarcinoma (5), squamous cell carcinoma (26), breast cancer (33), and hepatocellular carcinoma (34), we also observed constitutive activation of MEK and Erk in MG-63 cells as assessed by their phosphorylation. This activation might be the result of overexpression of protein kinase C isozymes in various malignant cells (6). Furthermore, constitutive activation of receptor tyrosine kinases, particularly c-Met, has also been reported in prolactin-secreting breast cancer (40) and renal carcinoma cells (27). Unlike the complete abolition of c-Met phosphorylation after HGF neutralization, MEK1 and Erk1/2 exhibited some basal phosphorylation. This result implies the existence of other factors responsible for their constitutive phosphorylation; however, our data demonstrate that HGF is one factor contributing to their phosphorylation in view of the dose- and time-dependent phosphorylation of MEK1 and its downstream Erk1/2 after addition of exogenous HGF. Our data showing that inhibition of endogenous HGF synthesis by 1,25(OH)2D3 significantly reduces the extent of constitutive phosphorylation of Erk1/2 further supports the concept that endogenous HGF contributes significantly to constitutive phosphorylation of Erk1/2, whereas the nonphosphorylated proteins remain unaltered. Participation of the MEK pathway in cellular proliferation has been observed in many other cell types, where it is activated by both protein kinase C-dependent (1, 20, 25, 30) and -independent mechanisms (4). Clearly, additional studies are required to understand the various mechanism(s) underlying the HGF-induced phosphorylation of these MAPKs.

Although our report strongly implicates the MEK-Erk pathway in HGF-induced mitogenesis of MG-63 cells, an unequivocal demonstration would require showing abolition of HGF-induced proliferation by inhibition of MEK with specific pharmacological inhibitors such as PD-98059 or U-0126. However, we were unable to perform such experiments because of the robust secretion of HGF by MG-63 cells, which over the several-hour time course of such an experiment would result in constitutive activation of the HGF-c-Met-MEK-Erk pathway.

Because 1,25(OH)2D3 inhibits cellular growth and induces differentiation of MG-63 cells to a more mature osteoblast-like phenotype (13, 32), we studied the role of this hormone on HGF synthesis. Our results show that 1,25(OH)2D3 profoundly inhibits both HGF synthesis and secretion. Because HGF is proproliferative in these cells, it is safe to say that 1,25(OH)2D3-mediated growth inhibition is achieved, at least in part, by inhibiting HGF synthesis. Only two reports have appeared examining the effect of 1,25(OH)2D3 on HGF synthesis [one showing stimulation and one demonstrating inhibition in chondrocytes (16) and HL-60 human promyelocytic leukemia cells (21)]. In the latter cell type, where 1,25(OH)2D3 promotes differentiation to a monocyte/macrophage phenotype, it has been shown that 1,25(OH)2D3 inhibits phorbol 12-myristate, 13-acetate-induced synthesis/secretion of HGF without affecting its basal rate of synthesis (19). However, in MG-63 cells, a model of osteoblastic differentiation, our study reveals that 1,25(OH)2D3 inhibits the constitutive synthesis of HGF, likely by acting at the transcriptional level, thereby demonstrating a cell type-specific difference in the effect of 1,25(OH)2D3 on HGF synthesis.

Recently, cortisol, a hormone that signals through a specific nuclear receptor, has been shown to inhibit HGF mRNA expression in rat calvarial osteoblasts (8) and in human osteoblast-like cells (36). Furthermore, we have shown that ligands of other nuclear receptors, such as retinoic acid receptors and RxRs, inhibit HGF mRNA expression in U-87 human astrocytoma cells (9). Because the VDR could exert its effect on transcription by heterodimerizing with RxRs, it is possible that in MG-63 cells RxR ligands could have additive effects on 1,25(OH)2D3-mediated downregulation of HGF mRNA.

HGF expression has been shown to be increased by fibroblast growth factor (FGF)-2 in the mouse calvarial osteoblast cell line, MC3T3-E1, and in rat primary calvarial osteoblasts (7). Interestingly, both cortisol and FGF increased the expression of c-Met in calvarial osteoblasts. In our study, 1,25(OH)2D3 at concentrations that inhibit HGF synthesis failed to induce any change in c-Met expression. Therefore, 1,25(OH)2D3 attenuates an HGF-mediated proliferative loop acting in an autocrine mode by reducing the HGF ligand but not its receptor. Consequently, the exact nature of the effect of 1,25(OH)2D3 on c-Met expression in MG-63 cells awaits molecular analysis of the c-Met promoter for the presence of a VDRE functional site(s).

In summary, we show the existence of an autocrine loop for HGF in MG-63 cells that supports mitogenesis, most likely because of c-Met-induced activation of the MEK/Erk pathway. Furthermore, 1,25(OH)2D3 inhibits proliferation of MG-63 cells by interfering with this autocrine loop by inhibiting HGF synthesis without any accompanying change in its receptor, c-Met. Our results suggest a new mechanism underlying 1,25(OH)2D3-mediated growth inhibition of MG-63 cells.


    ACKNOWLEDGEMENTS

Generous support was received from the following sources: a Pfizer/American Federation for Aging Research New Faculty Development Award and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-02215-01A2 (to N. Chattopadhyay), a Martin Brotman Advanced Training/Transition Award from the American Digestive Health Foundation (to R. J. MacLeod), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48330, DK-52005, and DK-41415, NPS Pharmaceuticals, and the St. Giles Foundation (to E. M. Brown).


    FOOTNOTES

Address for reprint requests and other correspondence: N. Chattopadhyay, Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115 (E-mail: Naibedya{at}rics.bwh.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 27, 2002;10.1152/ajpendo.00247.2002

Received 6 June 2002; accepted in final form 22 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abe, K, and Saito H. The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C. Neurosci Res 36: 251-257, 2000[ISI][Medline].

2.   Abounader, R, Ranganathan S, Kim BY, Nichols C, and Laterra J. Signaling pathways in the induction of c-met receptor expression by its ligand scatter factor/hepatocyte growth factor in human glioblastoma. J Neurochem 76: 1497-1508, 2001[ISI][Medline].

3.   Arihiro, K, and Inai K. Expression of CD31, Met/hepatocyte growth factor receptor and bone morphogenetic protein in bone metastasis of osteosarcoma. Pathol Int 51: 100-106, 2001[ISI][Medline].

4.   Awasthi, V, and King RJ. PKC, p42/p44 MAPK, and p38 MAPK are required for HGF-induced proliferation of H441 cells. Am J Physiol Lung Cell Mol Physiol 279: L942-L949, 2000[Abstract/Free Full Text].

5.   Bang, YJ, Kwon JH, Kang SH, Kim JW, and Yang YC. Increased MAPK activity and MKP-1 overexpression in human gastric adenocarcinoma. Biochem Biophys Res Commun 250: 43-47, 1998[ISI][Medline].

6.   Basu, A. The potential of protein kinase C as a target for anticancer treatment. Pharmacol Ther 59: 257-280, 1993[ISI][Medline].

7.   Blanquaert, F, Delany AM, and Canalis E. Fibroblast growth factor-2 induces hepatocyte growth factor/scatter factor expression in osteoblasts. Endocrinology 140: 1069-1074, 1999[Abstract/Free Full Text].

8.   Blanquaert, F, Pereira RC, and Canalis E. Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts. Am J Physiol Endocrinol Metab 278: E509-E515, 2000[Abstract/Free Full Text].

9.   Chattopadhyay, N, Butters RR, and Brown EM. Agonists of the retinoic acid- and retinoid X-receptors inhibit hepatocyte growth factor secretion and expression in U87 human astrocytoma cells. Brain Res Mol Brain Res 87: 100-108, 2001[ISI][Medline].

10.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

11.   Clover, J, and Gowen M. Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone 15: 585-591, 1994[ISI][Medline].

12.   Ferracini, R, Scotlandi K, Cagliero E, Acquarone F, Olivero M, Wunder J, and Baldini N. The expression of Met/hepatocyte growth factor receptor gene in giant cell tumors of bone and other benign musculoskeletal tumors. J Cell Physiol 184: 191-196, 2000[ISI][Medline].

13.   Finch, JL, Dusso AS, Pavlopoulos T, and Slatopolsky EA. Relative potencies of 1,25-(OH)(2)D(3) and 19-Nor-1,25-(OH)(2)D(2) on inducing differentiation and markers of bone formation in MG-63 cells. J Am Soc Nephrol 12: 1468-1474, 2001[Abstract/Free Full Text].

14.   Fukuda, T, Ichimura E, Shinozaki T, Sano T, Kashiwabara K, Oyama T, Nakajima T, and Nakamura T. Coexpression of HGF and c-Met/HGF receptor in human bone and soft tissue tumors. Pathol Int 48: 757-762, 1998[ISI][Medline].

15.   Grano, M, Galimi F, Zambonin G, Colucci S, Cottone E, Zallone AZ, and Comoglio PM. Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro. Proc Natl Acad Sci USA 93: 7644-7648, 1996[Abstract/Free Full Text].

16.   Grumbles, RM, Howell DS, Wenger L, Altman RD, Howard GA, and Roos BA. Hepatocyte growth factor and its actions in growth plate chondrocytes. Bone 19: 255-261, 1996[ISI][Medline].

17.   Haussler, MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, and Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325-349, 1998[ISI][Medline].

18.   Hjalm, G, MacLeod RJ, Kifor O, Chattopadhyay N, and Brown EM. Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase. J Biol Chem 276: 34880-34887, 2001[Abstract/Free Full Text].

19.   Hoshino, R, Tanimura S, Watanabe K, Kataoka T, and Kohno M. Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1). J Biol Chem 276: 2686-2692, 2001[Abstract/Free Full Text].

20.   Hussaini, IM, Karns LR, Vinton G, Carpenter JE, Redpath GT, Sando JJ, and VandenBerg SR. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells. J Biol Chem 275: 22348-22354, 2000[Abstract/Free Full Text].

21.   Inaba, M, Koyama H, Hino M, Okuno S, Terada M, Nishizawa Y, Nishino T, and Morii H. Regulation of release of hepatocyte growth factor from human promyelocytic leukemia cells, HL-60, by 1,25-dihydroxyvitamin D3, 12-O-tetradecanoylphorbol 13-acetate, and dibutyryl cyclic adenosine monophosphate. Blood 82: 53-59, 1993[Abstract].

22.   Jiang, WG, and Hiscox S. Hepatocyte growth factor/scatter factor, a cytokine playing multiple and converse roles. Histol Histopathol 12: 537-555, 1997[ISI][Medline].

23.   Jukkola, A, Risteli L, Melkko J, and Risteli J. Procollagen synthesis and extracellular matrix deposition in MG-63 osteosarcoma cells. J Bone Miner Res 8: 651-657, 1993[ISI][Medline].

24.   Kifor, O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, and Brown EM. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 280: F291-F302, 2001[Abstract/Free Full Text].

25.   Kolkova, K, Novitskaya V, Pedersen N, Berezin V, and Bock E. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway. J Neurosci 20: 2238-2246, 2000[Abstract/Free Full Text].

26.   Mishima, K, Yamada E, Masui K, Shimokawara T, Takayama K, Sugimura M, and Ichijima K. Overexpression of the ERK/MAP kinases in oral squamous cell carcinoma. Mol Pathol 11: 886-891, 1998.

27.   Nakamura, T, Kanda S, Yamamoto K, Kohno T, Maeda K, Matsuyama T, and Kanetake H. Increase in hepatocyte growth factor receptor tyrosine kinase activity in renal carcinoma cells is associated with increased motility partly through phosphoinositide 3-kinase activation. Oncogene 20: 7610-7623, 2001[ISI][Medline].

28.   Nakashiro, K, Okamoto M, Hayashi Y, and Oyasu R. Hepatocyte growth factor secreted by prostate-derived stromal cells stimulates growth of androgen-independent human prostatic carcinoma cells. Am J Pathol 157: 795-803, 2000[Abstract/Free Full Text].

29.   Naldini, L, Vigna E, Narsimhan RP, Gaudino G, Zarnegar R, Michalopoulos GK, and Comoglio PM. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 6: 501-514, 1991[ISI][Medline].

30.   Pae, HO, Yoo JC, Choi BM, Lee EJ, Song YS, and Chung HT. 12-O-tetradecanoyl phorbol 13-acetate, protein kinase C (PKC) activator, protects human leukemia HL-60 cells from taxol-induced apoptosis: possible role for extracellular signal-regulated kinase. Immunopharmacol Immunotoxicol 22: 61-73, 2000[ISI][Medline].

31.   Rachez, C, and Freedman LP. Mechanisms of gene regulation by vitamin D(3) receptor: a network of coactivator interactions. Gene 246: 9-21, 2000[ISI][Medline].

32.   Ryhanen, S, Jaaskelainen T, Saarela JT, and Maenpaa PH. Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093). J Cell Biochem 70: 414-424, 1998[ISI][Medline].

33.   Salh, B, Marotta A, Matthewson C, Ahluwalia M, Flint J, Owen D, and Pelech S. Investigation of the Mek-MAP kinase-Rsk pathway in human breast cancer. Anticancer Res 19: 731-740, 1999[ISI][Medline].

34.   Schmidt, CM, McKillop IH, Cahill PA, and Sitzmann JV. Increased MAPK expression and activity in primary human hepatocellular carcinoma. Biochem Biophys Res Commun 236: 54-58, 1997[ISI][Medline].

35.   Scotlandi, K, Baldini N, Oliviero M, Di Renzo MF, Martano M, Serra M, Manara MC, Comoglio PM, and Ferracini R. Expression of Met/hepatocyte growth factor receptor gene and malignant behavior of musculoskeletal tumors. Am J Pathol 149: 1209-1219, 1996[Abstract].

36.   Skrtic, S, and Ohlsson C. Cortisol decreases hepatocyte growth factor levels in human osteoblast-like cells. Calcif Tissue Int 66: 108-112, 2000[ISI][Medline].

37.   Taichman, R, Reilly M, Verma R, Ehrenman K, and Emerson S. Hepatocyte growth factor is secreted by osteoblasts and cooperatively permits the survival of haematopoietic progenitors. Br J Haematol 112: 438-448, 2001[ISI][Medline].

38.   Tomioka, D, Maehara N, Kuba K, Mizumoto K, Tanaka M, Matsumoto K, and Nakamura T. Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model. Cancer Res 61: 7518-7524, 2001[Abstract/Free Full Text].

39.   Wallenius, V, Hisaoka M, Helou K, Levan G, Mandahl N, Meis-Kindblom JM, Kindblom LG, and Jansson JO. Overexpression of the hepatocyte growth factor (HGF) receptor (Met), and presence of a truncated and activated intracellular HGF receptor fragment in locally aggressive/malignant human musculoskeletal tumors. Am J Pathol 156: 821-829, 2000[Abstract/Free Full Text].

40.   Yamauchi, T, Yamauchi N, Ueki K, Sugiyama T, Waki H, Miki H, Tobe K, Matsuda S, Tsushima T, Yamamoto T, Fujita T, Taketani Y, Fukayama M, Kimura S, Yazaki Y, Nagai R, and Kadowaki T. Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer. J Biol Chem 275: 33937-33944, 2000[Abstract/Free Full Text].

41.   Zambonin, G, Camerino C, Greco G, Patella V, Moretti B, and Grano M. Hydroxyapatite coated with hepatocyte growth factor (HGF) stimulates human osteoblasts in vitro. J Bone Joint Surg Br 82: 457-460, 2000[Medline].

42.   Zenmyo, M, Komiya S, Hamada T, Hiraoka K, Kato S, Fujii T, Yano H, Irie K, and Nagata K. Transcriptional activation of p21 by vitamin D(3) or vitamin K(2) leads to differentiation of p53-deficient MG-63 osteosarcoma cells. Hum Pathol 32: 410-416, 2001[ISI][Medline].


Am J Physiol Endocrinol Metab 284(1):E219-E227
0193-1849/03 $5.00 Copyright © 2003 the American Physiological Society