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Cell Biology International (2008) 32, 1126–1135 (Printed in Great Britain)
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Prolactin decreases the expression ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin in human fetal osteoblast cells
Dutmanee Seriwatanachaia, Narattaphol Charoenphandhuac*, Tuangporn Suthiphongchaibc and Nateetip Krishnamraac*
aDepartment of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
bDepartment of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
cConsortium for Calcium and Bone Research, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

Abstract

Prolactin (PRL) enhanced bone remodeling leading to net bone loss in adult and net bone gain in young animals. Studies in PRL-exposed osteoblasts derived from adult humans revealed an increase in the expression ratio of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), thus supporting the previous finding of PRL-induced bone loss in adults. This study thus investigated the effects of PRL on the osteoblast functions and the RANKL/OPG ratio in human fetal osteoblast (hFOB) cells which strongly expressed PRL receptors. After 48h incubation, PRL increased osteocalcin expression, but had no effect on cell proliferation. However, the alkaline phosphatase activity was decreased in a dose–response manner within 24h. The effect of PRL on alkaline phosphatase was abolished by LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. PRL also decreased the RANKL/OPG ratio by downregulating RANKL and upregulating OPG expression, implicating a reduction in the osteoblast signal for osteoclastic bone resorption. It could be concluded that, unlike the osteoblasts derived from adult humans, PRL-exposed hFOB cells exhibited indices suggestive of bone gain, which could explain the in vivo findings in young rats. The signal transduction of PRL in osteoblasts involved the PI3K pathway.

Keywords: Alkaline phosphatase, hFOB, OPG, Osteocalcin, PI3K, Prolactin receptor, RANKL.

*Corresponding authors. Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Tel./fax: +66 2354 7154.

1 Introduction

Pregnant and lactating mammals use prolactin (PRL) as a calcium-regulating hormone to stimulate intestinal calcium absorption and mobilize calcium from bone for fetal development and milk production (Charoenphandhu and Krishnamra, 2007; Lotinun et al., 1998; Thongon et al., 2008). However, PRL action on calcium metabolism was also reported in non-pregnant/lactating rats, in which PRL induced a positive calcium balance by directly stimulating the intestinal calcium absorption and renal calcium reabsorption (Jantarajit et al., 2007; Piyabhan et al., 2000). Interestingly, young rats were more responsive to PRL than adult and aging rats (Krishnamra et al., 1993; Krishnamra and Seemoung, 1996).

Studies of the in vivo effects of PRL on bone are generally complicated by chronic estrogen deficiency caused by PRL-induced hypogonadism (Wang and Chan, 1982; Wang et al., 1980). However, osteoblasts have been known to express PRL receptors (PRLR), which indicated that bone cells are direct targets of PRL (Coss et al., 2000). Our in vivo studies using bone histomorphometry in adult rats showed that PRL exerted an estrogen-independent action by enhancing bone turnover with a greater effect on bone resorption (Seriwatanachai et al., 2008). At the cellular level, PRL directly decreased osteocalcin expression and alkaline phosphatase activity in an osteoblast cell line (MG-63) derived from an adult human, thus supporting the in vivo findings of PRL-induced bone loss (Seriwatanachai et al., 2008). Interestingly, the effects of PRL on bone varied with age. In contrast to adult rats (more than 8weeks old), PRL stimulated calcium deposition and induced net bone gain in femur, tibia and sternum of 3-week-old young rats (Krishnamra and Seemoung, 1996). We therefore hypothesized that, unlike its action in MG-63 osteoblasts derived from adult humans, PRL may increase the cellular activities of osteoblasts derived from young humans leading to bone formation. Although the expression of PRLR in human fetal osteoblast (hFOB) cell lines had not been reported, we herein used differentiated hFOB cells, which had minimal chromosome aberration and exhibited the matrix-producing properties of normal differentiated osteoblasts (Harris et al., 1995; Subramaniam et al., 2002), in the investigation of PRL actions. In addition, undifferentiated hFOB cells have recently been shown to possess multilineage differentiation potential (Yen et al., 2007), suggesting that they retained the characteristics of fetal cells.

Bone turnover is a coupled process of the osteoblastic bone formation and osteoclastic bone resorption. Since osteoclasts did not express PRLR (Coss et al., 2000; Kelly et al., 2001), enhanced bone resorption in hyperprolactinemic rats could be due to changes in the osteoblast-expressed mediators, the receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG). Binding of RANKL to its receptors on osteoclasts stimulated bone resorption, whereas binding to its decoy receptors, OPG, decreased bone resorption (Kostenuik, 2005). Thus, the RANKL/OPG ratio determined osteoclast activity, bone resorption as well as bone turnover (Abdallah et al., 2005; Grimaud et al., 2003; Kostenuik, 2005). Our recent findings of the PRL-induced increase in the RANKL/OPG ratio in MG-63 cells and decrease in the OPG expression in primary osteoblasts from adult rats supported the in vivo report of net bone loss in adult hyperprolactinemic rats (Seriwatanachai et al., 2008). Hence, it was possible that hFOB cells may respond to PRL by decreasing the RANKL/OPG expression ratio.

Nothing is known regarding PRL signaling in osteoblasts. The putative signaling pathway of PRL in mammary epithelia was the Janus kinase (JAK2) pathway (Bole-Feysot et al., 1998), whereas the phosphoinositide 3-kinase (PI3K) pathway was reported in non-mammary tissues, e.g., liver, duodenum, colon, pancreatic islets, and Nb2 lymphoma cells (Amaral et al., 2004; Bishop et al., 2006; Jantarajit et al., 2007; Puntheeranurak et al., 2007; Yamauchi et al., 1998). We recently demonstrated that the PRL-stimulated transepithelial calcium transport in the duodenum was via the PI3K, and not the JAK2 pathway (Jantarajit et al., 2007). Therefore, signal transduction of PRL in osteoblasts may also occur via the PI3K.

The objectives of the present study were (i) to demonstrate the expression of PRLR in hFOB cells; (ii) to study the effect of PRL on functions of hFOB cells, including cell proliferation, osteocalcin expression, and alkaline phosphatase activity; (iii) to show whether there was a change in the expression ratio of RANKL/OPG in PRL-exposed hFOB cells; and (iv) to investigate whether PRL signaling in hFOB cells involved the PI3K pathway.

2 Materials and methods

2.1 Cell culture

Human fetal osteoblast 1.19 (hFOB) cells (ATCC No. CRL-11372), an immortalized cell line, were propagated in DMEM/F-12 media, supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin/streptomycin, and 0.25μL/mL amphotericin B (Sigma, St. Louis, MO, USA). Cells were cultured in 75-cm2 T-flasks at 37°C in a 5% CO2 in air humidified incubator. Culture medium was changed every 2days, and the cultures were split 1:10 when cells had reached 80% confluence. Cells were counted using a hemocytometer and trypan blue dye exclusion.

Osteoblast-like MG-63 cells (ATCC No. CRL-1427; a kind gift from Dr Suttatip Kamolmatyakul, Prince of Songkla University, Thailand), derived from human osteosarcoma, were cultured in 75-cm2 T-flasks with α-MEM supplemented with 10% FBS, 100U/mL penicillin/streptomycin, and 0.25μL/mL amphotericin B (Sigma). To induce maximal expression of PRLR, 1μM dexamethasone and 0.1μM 1,25-(OH)2D3 (Sigma) were also added to the medium, as previously described (Bataille-Simoneau et al., 1996). Cells were incubated at 37°C with 5% CO2, and subcultured according to the ATCC's protocol.

2.2 Immunofluorescent analysis

hFOB cells were cultured on a coverslip at 105 cells/coverslip in the presence of 0.2% FBS for 16h. Cells were fixed for 10min with 3% paraformaldehyde and 2% sucrose at 25°C, washed 3 times with PBS, and permeabilized for 5min with 0.5% Triton X-100 in PBS at 25°C. Non-specific sites were blocked with 10% FBS for 30min at room temperature. Samples were then incubated overnight at 4°C with 1:300 rabbit polyclonal anti-PRLR primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally with 1:200 Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA). Images were captured with an inverted fluorescent microscope (model Eclipse TE2000-U; Nikon, Tokyo, Japan).

2.3 Cell proliferation assay

hFOB cells were inoculated in a 96-well culture plate (5000cells/well). After 48h incubation with 1, 10, 100, 1000ng/mL PRL, the culture medium was replaced by a medium containing 10% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). After 3h incubation with MTT at 37°C, the absorbance of each well was determined at 540nm by a microplate reader (model Multiscan EX; Thermo Labsystems, Cergy-Pontoise, France), as described previously (Mosmann, 1983). The absorbance of control cells was considered to be 100%. The relative proliferation of PRL-exposed cells was presented as a percent of control. Each sample was an average of 6 replications, and the relative proliferation of each n was averaged from 3 independent samples (triplicate).

2.4 Alkaline phosphatase activity assay

MG-63 or hFOB cells were cultured in 6-well culture plates (105cells/well). Alkaline phosphatase activity was determined by the conversion of p-nitrophenyl phosphate to p-nitrophenol, as previously described (Coss et al., 2000). In brief, cells were washed twice with PBS pH 7.4, and incubated for 1h with 2mL solution containing (in mM) 100 Na2CO3, 10 MgCl2, 20 p-nitrophenyl phosphate (Sigma), pH 10.3. Thereafter, 1mL of 5M NaOH was added. Color development was quantified immediately at 410nm.

2.5 Preparation of total RNA and RT-PCR

As previously described (Charoenphandhu et al., 2007; Seriwatanachai et al., 2008), the total RNA of hFOB cells was prepared by using the RNeasy mini kit (Qiagen, Valencia, CA, USA). Two micrograms of the total RNA were reverse-transcribed with the oligo-dT20 primer and SuperScript III kit (Invitrogen, Carlsbad, CA, USA) to cDNA by a thermal cycler (model Minicycler; MJ Research, Watertown, MA, USA). Sense and antisense primers for PRLR, osteocalcin, RANKL, OPG, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown in Table 1. GAPDH served as a control gene to check the consistency of the reverse transcription and to normalize values between samples. After amplification with Taq polymerase (Qiagen), the PCR products were visualized on a 1% agarose gel stained with 1.0μg/mL ethidium bromide under a trans-UV system (model Quantity One 2000; BioRad, Hercules, CA, USA). The cycle-band intensity curve was plotted for each gene to obtain an optimal PCR cycle which fell in the exponential phase. For a semi-quantitative analysis, the expression of a studied gene in the control group was considered to be 100%, while that in the experimental group was calculated as a percent change relative to the value of the control group.

Table 1.

Homo sapiens oligonucleotide sequences used in the PCR experiment

GeneReference or accession no.Primer (forward/reverse)Product length (bp)Cycle
PRLRa5′-AAATGTGGCATCTGCAACCGTTTTCAC-3′179030
5′-GCACTTGCTTGATGTTGCAGTGAAGTT-3′
OCa5′-GGCCAGGCAGGTGCGAAGC-3′27130
5′-GCCAGGCCAGCAGAGCGACAC-3′
RANKLa5′-GCCAGTGGGAGATGTTAG-3′48633
5′-TTAGCTGCAAGTTTTCCC-3′
OPGa5′-GCTAACCTCACCTTCGAG-3′32422
5′-TGATTGGACCTGGTTACC-3′
GAPDHbNM_0020465′-CACCCACTCCTCCACCTTTG-3′11020
5′-CCACCACCCTGTTGCTGTAG-3′
a Seriwatanachai et al., 2008.
b Custom-design primer.

2.6 Western blot analysis

hFOB cells were lysed in a lysis buffer (150mM Tris–HCl pH 7.4, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 1% Nonidet P-40, protease inhibitor, 1M NaF, 1M β-glycerophosphate, 0.5M Na3VO4, 1M DTT, 1% sodium deoxycholate, and 5mM EDTA) (Sigma). After 30min incubation at 4°C, lysates were sonicated and centrifuged at 12,000×g for 10min at 4°C, and heated for 5min at 95°C before being loaded on a gel. Proteins (100μg/well) were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK) by electroblotting. Non-specific binding sites on the membrane were blocked for 1h at room temperature by 5% skim milk in TBS containing 0.1% Tween-20. Membranes were probed overnight at 4°C with 1:2000 rabbit polyclonal anti-RANKL or anti-OPG primary antibodies (Santa Cruz), and re-probed with 1:5000 mouse anti-β-actin antibody (Santa Cruz). After 1h incubation at 25°C with 1:2000 goat anti-rabbit or anti-mouse secondary antibodies (Santa Cruz), blots were visualized using the enhanced chemiluminescence (ECL Plus) kit (Amersham). Expression of each protein in the control group was considered as 100%.

2.7 Experimental protocols Protocol 1

The objective of this protocol was to determine the expression of PRLR in hFOB cells. Normally, osteoblasts constitutively express PRLR; however, some osteoblastic cell lines, e.g., MG-63 cells, require mediators such as vitamin D or dexamethasone for PRLR expression (Bataille-Simoneau et al., 1996). Therefore, hFOB cells were cultured in the presence of 0.2% and 10% fetal bovine serum (FBS; controls), 0.1μM 1,25-(OH)2D3 (Vit D) (Sigma), 1μM dexamethasone (DEX) (Sigma), or a combination of Vit D and DEX. Expression of PRLR transcripts was determined by RT-PCR, and expression of PRLR proteins was demonstrated by immunofluorescent imaging.

Protocol 2

To investigate the direct effects of PRL on osteoblast functions, hFOB cells were incubated in normal media (control) or medium containing 1, 10, 100 or 1000ng/mL recombinant human PRL (rhPRL) (purity >97% as determined by SDS-PAGE; R&D Systems, Minneapolis, MN, USA) at 37°C for 0, 0.5, 3, 6, 12, 24 and 48h prior to determination of alkaline phosphatase activity. The maximal suppression of alkaline phosphatase activity by PRL was seen at 48h similar to that reported previously by Seriwatanachai et al. (2008). Therefore, the 48h incubation period was used to demonstrate the effects of PRL on osteoblast proliferation and functions, including osteocalcin mRNA expression and RANKL/OPG mRNA and protein expression. Expression of mRNAs and proteins was determined in triplicate by RT-PCR and Western blot analysis, respectively.

Protocol 3

Since one of the signaling pathways of PRL was the PI3K pathway (Jantarajit et al., 2007; Puntheeranurak et al., 2007), this protocol aimed to demonstrate whether PRL affected osteoblast activity via this pathway. hFOB or MG-63 cells were incubated at 37°C for 48h with normal media (control) or media containing 0.3% dimethyl sulfoxide (DMSO; vehicle; Sigma), 100nM LY294002 (PI3K inhibitor; Sigma), 100ng/mL rhPRL, or 100ng/mL rhPRL plus 100nM LY294002. Finally, the alkaline phosphatase activity was determined.

2.8 Statistical analysis

Results are expressed as mean±SEM. Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Newman–Keuls post-test. The level of significance for all statistical tests was P<0.05. Data were analyzed by GraphPad Prism 4.0 for Mac OS X (GraphPad Software, San Diego, CA, USA).

3 Results

3.1 hFOB cells expressed mRNAs and proteins of PRLR

Our RT-PCR study revealed a constitutive expression of PRLR in hFOB cells under the control condition (Fig. 1A). In contrast to the previous report on MG-63 cells (Seriwatanachai et al., 2008), the expression of PRLR in hFOB cells was not altered by 1,25-(OH)2D3, dexamethasone or the combination of both. Both agents were known to upregulate PRLR in other osteoblast cell lines, e.g., MG-63 and Saos-2 cells (Bataille-Simoneau et al., 1996). Immunofluorescent analysis confirmed that hFOB cells strongly expressed PRLR proteins (Fig. 1B). These findings indicated that this fetal osteoblast cell line served as a target of PRL.

Fig. 1

(A) Expression of PRLR transcripts in hFOB cells exposed for 48h to 0.2% and 10% fetal bovine serum (FBS, control), 0.1μM 1,25-(OH)2D3 (Vit D), 1μM dexamethasone (DEX), or a combination of Vit D and DEX (Vit D+DEX). A representative electrophoretic image of PRLR is also demonstrated. Numbers in parentheses are numbers of independent flasks. (B) A representative immunofluorescent image showing PRLR protein expression in hFOB cells (n=5), magnification ×400.

3.2 PRL upregulated osteocalcin expression in hFOB cells

Since PRL stimulates bone growth and bone calcium deposition in young animals, we studied osteoblast functions that were associated with bone formation. We found that PRL had no effect on hFOB cell proliferation (Fig. 2A). However, 100 and 1000ng/mL rhPRL increased the expression of osteocalcin mRNA from the control level (100.00±5.74%) to 155.27±13.02% (n=5, P<0.05) and 174.73±12.08 (n=5, P<0.01), respectively. The results implied the stimulatory effect of PRL on osteoblastic functions in fetal osteoblasts.

Fig. 2

Dose-dependent changes in (A) cell proliferation and (B) osteocalcin mRNA expression in hFOB cells incubated for 48h with 1, 10, 100 or 1000ng/mL rhPRL. Values of the control groups were normalized to 100%. *P<0.05, **P<0.01 compared with the control group. Numbers in parentheses are numbers of independent flasks. Experiments were performed in triplicate.

3.3 PRL decreased alkaline phosphatase activity in hFOB cells

In contrast to the osteocalcin expression, the activity of alkaline phosphatase was decreased by 100 and 1000ng/mL rhPRL to 83.31±2.97% (n=5, P<0.05) and 77.70±5.07% (n=5, P<0.01), respectively (Fig. 3A). A time-dependent study showed a significant decrease in alkaline phosphatase activity at 24h after exposure to 1000ng/mL rhPRL (Fig. 3B).

Fig. 3

Dose-dependent changes in alkaline phosphatase activity (A) in hFOB cells incubated for 48h with 1, 10, 100 or 1000ng/mL rhPRL. Values of the control groups were normalized to 100%. *P<0.05, **P<0.01 compared with the control group. (B) Time-dependent changes in alkaline phosphatase activity in hFOB cells exposed to 1000ng/mL rhPRL. The value at 0h was normalized to 100%. †††P<0.001 compared with the values at 0h. Numbers in parentheses are numbers of independent flasks. Experiments were performed in triplicate.

3.4 PRL decreased the expression ratio of RANKL/OPG in hFOB cells

Effects of rhPRL exposure on the markers of osteoblast-mediated activation of bone resorption are presented in Fig. 4. The expression ratio of RANKL and OPG, both of which were synthesized by osteoblasts, represented bone resorption (Kostenuik, 2005). Expression of RANKL transcripts were decreased by 10, 100 and 1000ng/mL rhPRL to 76.56±4.97% (n=3, P<0.05), 76.81±3.96% (n=3, P<0.05) and 57.58±7.73% (n=3, P<0.01) of the control level, respectively, while RANKL protein was decreased by 100 and 1000ng/mL rhPRL to 71.53±2.79% (n=7, P<0.01) and 64.78±6.46% (n=7, P<0.01). Although PRL did not change the expression of OPG transcripts, 100 and 1000ng/mL rhPRL upregulated expression of OPG protein to 122.96±5.42% (n=5, P<0.05) and 140.12±7.53% (n=5, P<0.001), respectively. Therefore, the ratios of RANKL/OPG mRNAs in hFOB cells were significantly decreased by 24, 21 and 43% after 48h exposure to 10, 100 and 1000ng/mL rhPRL, respectively, while the ratios of RANKL/OPG proteins were decreased by 41% and 56% after 100 and 1000ng/mL rhPRL exposure. The decreased RANKL/OPG ratio implicated a suppression of the osteoclast-mediated bone resorption by PRL.

Fig. 4

Dose-dependent changes in the mRNA expressions of OPG (A) and RANKL (B), protein expressions of OPG (C) and RANKL (D), and the ratios of RANKL/OPG mRNA (E) and protein (F) expressions in hFOB cells incubated for 48h with 1, 10, 100 or 1000ng/mL rhPRL. Values of the control groups were normalized to 100%. *P<0.05, **P<0.01, ***P<0.001 compared with the control group. Numbers in parentheses are numbers of independent flasks. Experiments were performed in triplicate.

3.5 PRL-mediated decreases in alkaline phosphatase activity in hFOB and MG-63 cells were completely blocked by a PI3K inhibitor

Because the PI3K pathway was one of the signaling pathways of PRL, we investigated whether PRL used this pathway in osteoblasts (i.e., hFOB and MG-63 cells). We found that LY294002, a specific PI3K inhibitor, at concentrations ranging from 10nM to 1μM did not affect the viability or the proliferation rate of hFOB and MG-63 cells (Fig. 5A,B). Exposure to 100ng/mL rhPRL decreased alkaline phosphatase activity in both hFOB and MG-63 cells; however, this PRL action was completely abolished by 100nM LY294002 (Fig. 5C,D). DMSO, a vehicle for LY294002 preparation, and 100nM LY294002 alone had no effect on the alkaline phosphatase activity in both cell lines. The results suggested that PRL decreased the osteoblastic alkaline phosphatase activity via the PI3K pathway.

Fig. 5

(A,B) Proliferation of hFOB and MG-63 cells after incubation for 48h with 10nM, 100nM, 1μM or 10μM LY294002 (LY, PI3K inhibitor). (C,D) Alkaline phosphatase activity in hFOB and MG-63 cells incubated for 48h in normal culture media (control) or media containing 0.3% DMSO (vehicle), 100nM LY294002, 100ng/mL rhPRL, or 100ng/mL rhPRL plus 100nM LY294002. Values of the control groups were normalized to 100%. **P<0.01, ***P<0.001 compared with the control group. Numbers in parentheses are numbers of independent flasks. Experiments were performed in triplicate.

4 Discussion

In adult animals, high bone turnover is a characteristic of both physiological and pathological hyperprolactinemia (Krishnamra et al., 1997; Lotinun et al., 2003; Meaney et al., 2004). Generally, high bone turnover accelerates bone loss, especially when the resorption cavities are incompletely replaced. However, under certain conditions, such as during growth hormone administration, the increased bone turnover shifts the balance between bone formation and resorption toward net bone calcium deposition (Parfitt, 1991). Although high physiological PRL of &007E;75–100ng/mL during pregnancy did not produce a significant bone loss, transient osteopenia was reported after 3months of lactation when plasma PRL ranged between 200 and 350ng/mL (Prentice, 2000; Ritchie et al., 1998). Furthermore, high PRL levels (up to &007E;1000ng/mL) found in several pathological conditions, e.g., prolactinomas or prolonged antipsychotic drug use, induced massive bone loss and overt osteopenia (Biller et al., 1992; Crosignani, 2006; Meaney et al., 2004). Thus, PRL exposure in adult animals and humans, depending on the PRL concentrations, could lead to bone loss. On the other hand, by using the in vivo 45Ca kinetic study, Krishnamra and Seemoung (1996) reported that young rats responded differently to PRL, i.e., by enhancing bone gain instead of bone loss despite the presence of high bone turnover. Moreover, PRL administration in 3-week-old weaned rats resulted in a dose-dependent increase in the calcium content of femur, tibia and vertebrae (Krishnamra and Seemoung, 1996). The present investigation showed an increase in osteocalcin expression and a decrease in the RANKL/OPG ratio in hFOB cells which indicated that PRL-exposed osteoblasts derived from immature animals could potentially induce bone gain.

Indeed, the in vivo osteopenic action of PRL had long been explained by estrogen deficiency due to PRL-induced hypogonadism (Meaney et al., 2004; Wang et al., 1980). However, the PRLR knockout mice manifested a 60% decrease in the rate of bone formation (Clément-Lacroix et al., 1999), and PRL-exposed rats exhibited high bone turnover with different histomorphometric patterns from those seen in ovariectomized (Ovx) rats, i.e., higher mineral apposition rate and bone formation rate (Seriwatanachai et al., 2008). It is possible that PRL could also exert a direct estrogen-independent action on bone cells. The finding of PRLR in osteoblasts also supported this hypothesis. Although the levels of PRLR transcript in MG-63 cells were significantly elevated in the presence of 1,25-(OH)2D3 and dexamethasone (Bataille-Simoneau et al., 1996), the expression of PRLR mRNAs and proteins in hFOB cells similar to that in the primary rat osteoblasts (Seriwatanachai et al., 2008) was constitutive and independent of both hormones.

We further investigated the effect of PRL on hFOB cell functions and found that PRL stimulated osteocalcin expression in hFOB cells without affecting cell proliferation. This effect of PRL agreed with the recent report on neonatal osteoblasts (Seriwatanachai et al., 2008), and was also consistent with the action of other hormones, such as leptin which enhanced osteoblast differentiation but not proliferation (Thomas et al., 1999). On the other hand, MG-63 cells derived from adult humans showed a decrease in osteocalcin expression after a 48-h PRL exposure (Seriwatanachai et al., 2008). The PRL-induced increase in the activity of hFOB cells supported our hypothesis that PRL could increase bone formation in osteoblasts derived from young animals.

Similar to the primary neonatal rat osteoblasts (Coss et al., 2000) and MG-63 cells (Seriwatanachai et al., 2008), PRL-exposed hFOB cells manifested a decrease in alkaline phosphatase activity. Although alkaline phosphatase is a classical marker of bone formation (Stein et al., 1996), its expression depends on the developmental stage of osteoblasts (Owen et al., 1990). Normally, osteoblasts have roles in all 3 steps of bone formation, i.e., cell proliferation, extracellular matrix maturation, and mineralization (Owen et al., 1990). Responses of osteoblast proliferation, gene expression and enzyme activities to various humoral factors depend on the stage of development of the cells. For example, transforming growth factor β and its downstream protein Smad3 inhibited osteoblast proliferation, but enhanced alkaline phosphatase activity, mineralization, and expression of bone matrix proteins (Sowa et al., 2002). Generally, alkaline phosphatase expression is increased immediately after cessation of cell proliferation, while the expression of osteocalcin, which is important for the formation of hydroxyapatite crystal lattices (Hoang et al., 2003), is increased later during matrix maturation near the onset of mineralization (Owen et al., 1990). Therefore, a disparate relationship between osteocalcin expression and alkaline phosphatase activity could be observed during the development of osteoblasts.

Since PRL enhanced osteocalcin expression in the matrix maturation step (Fig. 2B) without affecting the in vitro mineralization of primary rat osteoblasts (Charoenphandhu et al., 2008), it appeared that PRL increased bone calcium deposition in young rats by downregulating RANKL and upregulating OPG, thereby decreasing the RANKL/OPG ratio. Similar to PRL, growth hormone which increases bone turnover and bone gain (Brixen et al., 2000; Landin-Wilhelmsen et al., 2003; Schlemmer et al., 1991) also stimulates OPG synthesis in hFOB cells (Mrak et al., 2007). In contrast, MG-63 cells responded to 48-h PRL exposure by increasing the RANKL/OPG ratio (Seriwatanachai et al., 2008). An increase in this ratio has been associated with osteopenic conditions, such as hyperparathyroidism and aging (Cao et al., 2003; Stilgren et al., 2004). In the transgenic mice, overexpression of RANKL increased the cortical porosity, whereas overexpression of OPG prevented bone loss and improved cortical bone strength (Kostenuik, 2005; Mizuno et al., 2002). The &007E;50% decrease in RANKL/OPG ratio in the present study implied that PRL could potentially suppress bone resorption, thus supporting the earlier report of greater calcium deposition in bones of PRL-treated young rats (Krishnamra and Seemoung, 1996).

Although the direct actions of PRL in osteoblasts have been demonstrated, nothing was known regarding its signaling pathway. PRL binding to PRLRs triggers dimerization of PRLRs and activation of the downstream signals (Bole-Feysot et al., 1998). In the mammary epithelia, PRL-PRLR complex used the JAK2 signaling pathway in the stimulation of milk production (Bole-Feysot et al., 1998). However, in other tissues, such as liver and calcium-transporting epithelia, e.g., duodenum and colon, mitogen-activated protein kinase (MAPK) and PI3K pathways have been reported (Amaral et al., 2004; Yamauchi et al., 1998). We recently showed that PRL directly stimulated duodenal calcium absorption (Jantarajit et al., 2007), and inhibited colonic Ca2+-dependent Cl− and K+ secretion via the PI3K pathway (Puntheeranurak et al., 2007). By using a potent inhibitor of PI3K (LY294002), the present study showed that the suppressive effect of PRL on alkaline phosphatase activity in hFOB cells was completely abolished. Therefore, the PI3K pathway could be one of the signaling pathways of PRL in osteoblasts. The detailed signaling cascade, however, remains to be investigated.

It can be concluded that hFOB cells strongly and constitutively express PRLR. PRL directly increases osteocalcin mRNA expression, and decreases the RANKL/OPG ratio in these cells, indicating the stimulation of bone formation and suppression of bone resorption, respectively. PRL also decreases alkaline phosphatase activity, in part, via the PI3K signaling pathway. Our in vitro study supported the previous in vivo findings that, unlike mature rats, PRL enhances bone calcium deposition and bone gain in young rats.

Acknowledgements

We thank Dr Sinee Disthabanchong from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, for her technical guidance and helpful comments. We thank Dr Suttatip Kamolmatyakul from the Prince of Songkla University, Thailand, for a kind gift of MG-63 cells. This research was supported by grants from the Royal Golden Jubilee Program (to DS), the Thailand Research Fund (TRF) and the National Center for Genetic Engineering and Biotechnology (BIOTEC).

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Received 18 December 2007/28 March 2008; accepted 30 April 2008

doi:10.1016/j.cellbi.2008.04.026
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