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Cell Biology International (2008) 32, 11261135 (Printed in Great Britain)
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
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 48
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.
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 8
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), 100
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, 100
2.2 Immunofluorescent analysis
hFOB cells were cultured on a coverslip at 105 cells/coverslip in the presence of 0.2% FBS for 16
2.3 Cell proliferation assay
hFOB cells were inoculated in a 96-well culture plate (5000
2.4 Alkaline phosphatase activity assay
MG-63 or hFOB cells were cultured in 6-well culture plates (105
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-dT
Homo sapiens oligonucleotide sequences used in the PCR experiment
2.6 Western blot analysis
hFOB cells were lysed in a lysis buffer (150
2.7 Experimental protocols 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), 0.1
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), 0.1
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 1000
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
2.8 Statistical analysis
Results are expressed as mean
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)
(A) Expression of PRLR transcripts in hFOB cells exposed for 48
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 1000
Dose-dependent changes in (A) cell proliferation and (B) osteocalcin mRNA expression in hFOB cells incubated for 48
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 1000
Dose-dependent changes in alkaline phosphatase activity (A) in hFOB cells incubated for 48
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 1000
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 48
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 10
(A,B) Proliferation of hFOB and MG-63 cells after incubation for 48
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;75–100
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)
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.
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 2008doi:10.1016/j.cellbi.2008.04.026