The effects of Femarelle and Vitamin D Analog on Lipoxygenase Products and Reactive Oxygen Species (ROS) formation in the Female and Male Human-Derived Cultured Osteoblastic Cells

Abstract

We have previously reported that human primary cultured bone cells (hObs) respond to estradiol-17β (E₂) and different phytoestrogens and vitamin D analogs by modulating cell proliferation. In the present study hObs derived from biopsies of pre- or post- menopausal females or males were cultured and treated with different estrogens and with the non-calcemic vitamin D JK-1624F₂-2 (JKF) and assayed for the expression of 12LO and 15LO mRNA as well as for the synthesis of 12 and 15HETEs and the formation of ROS. All cells express 12LO and 15LO and produce 12 and 15 HETEs. Treatment with all estrogens: E₂, daidzein (D) and femarelle (F) as well as JKF modulated the expression of both 12 and 15LO mRNA in pre- or post-menopausal female hObs and to some extent in male hObs. 12 and 15HETEs production was also modulated by these compounds in these cells. ROS formation was increased by all hormones in pre- and post- but not in male hObs. ROS formation was also increased by 12 and 15HETEs in pre- and post- but not in male hObs. In conclusion, we provide herein evidence for a novel mechanism of hormonal-induced human bone biology operating through LO enzymes and ROS formation, but the exact role of LO/HETE/ROS pathway is not clear yet. We also provide a mechanism of new phytoestrogen which is not behaving like E₂ and can react as both estrogenic compound and a disruptor of estrogen regulation in a mechanism which has still to be further studied.

Key words:

• estrogens
• JKF
• human osteoblasts
• lipoxygenase
• HETEs
• ROS

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2. Introduction

Estrogens appear to confer multiple skeletal protective effects in vitro (1), and in vivo (2,3), and to positively affect skeleton outcome in post- menopausal women (4). We found that vitamin D metabolites and analogs (5) as well as gonadal steroids and phytoestrogens stimulate DNA and CK as well as lipoxygenase (LO) mRNA and their products the HETEs formation (6). In the present study we analyzed the response of primary hObs derived from bone biopsies of pre- and post-menopausal female and male to estrogens, and to the vitamin D less calcemic analog JKF on the expression of LOs mRNA as well as HETEs production and reactive oxygen species (ROS) formation.

We focused on these enzymes in osteoblasts since LO products were shown to modulate MAPK activity (7,8) and proliferation or survival (9) in a number of cell types and could thus play a role in promoting cell growth. Additionally, there is a recent strong circumstantial evidence linking LO expression to bone mineral content (9,10). We hypothesized that the growth promoting effects of these hormones in human osteoblast-like cells may be associated with accelerated production of LO metabolites, whose putative action may explain some of the newly found links between LO and bone density.

Here we studied the modulation of LO mRNA expression and activity through the formation of 12- and 15- hydroxyeicosatetraenoic acid (HETE), the arachidonic acid derived metabolites of these enzymes as well as ROS production by estrogens and the vitamin D analog. We focused on these enzymes since LO products (8) to were shown to induce ROS formation (11), proliferation or survival, playing a role in promoting cell growth (12). We hypothesized that the growth modulating effects of the different compounds in human hObs might be associated with accelerated production of LO metabolites, whose putative action including effects on ROS production may explain some of the new links between the LO system and other biological activities (9).

Phytoestrogens are heterogeneous group of plant-derived compounds some of which are SERMs. All phytoestrogens are polyphenolic compounds with structural similarities to natural and synthetic estrogens; however they bind to the estrogen receptors with much lower affinity than E₂ (13-15). Soybeans and soy foods are the most significant dietary sources of the isoflavone class of phytoestrogens, which includes genisteine, daidzein and biochainin A (13) and have estrogenic activity in bone and other tissues and some anti estrogenic effect in the breast (14). Femarelle (F) is a unique enzymatic isolate of soybeans. F has some behaviour like a SERM when applied together with E2 and is stimulating bone cells (16-19). The present study focused on the interaction of F and other estrogens in bone cells in order to examine its beneficial and harm full effects of this new phytoestrogen with/without vitamin D analog in human derived osteoblasts in culture.

3. Results

3.1. Expression of 12 and 15LO in hObs

To investigate the basal levels of LO mRNA and HETEs in hObs, female-derived bone cells from both ages as well as male hObs expressed mRNA for both 12LO and 15LO as measured by real time PCR, corrected for RNAse P mRNA (12LO 0.066 in pre-, 0.013 in post- and 0.005 2^−δCT in male hObs; 15LO 1.2+0.1 in pre-, 2.0+0.13 in post- and 1.07+0.1 2^−δCT in male hObs (fig 1). These data suggest differences in the basal levels of both LO and its products.

Fig 1: The expression of 12 and 15LO mRNA and the production of 12 and 15 HETEs in cultured pre- and post- menopausal female and male- hObs. Details are given in the experimental section. Number of specimen n= 5 for each group.

3.2. Modulation by JKF, F, D or E₂ of 12 and 15LO in hObs

To investigate the hormonal responses of hObs, female-derived bone cells from both ages as well as male hObs which expressed mRNA for both 12LO and 15LO were treated with the different estrogens: E₂, D and F as well as JKF modulated the expression of 12 and 15LO to different extents in both female age groups and also by some compounds to some extent in male hObs (fig 2). These data suggest differences in the hormonal response of both LO mRNA expression.

Fig 2: The effects of treatment with E₂, D, F or JKF on 12 (upper panel) and 15 (lower panel) LO mRNA expression in pre-, post- menopausal and male- hObs. Cells were incubated for 3 days for mRNA expressions with F (20µg/ml), D (300 nM) or E₂ (3 nM) or JKF (1 nM). Results are expressed as % change in the 12 and 15LO mRNA expression quantified by real time PCR (n=4–8). *P<0.05; **p<0.01, compared with control incubates containing the vehicle for the active compound only.

3.3. Basal levels of 12 and 15HETEs production in hObs

To investigate the basal levels of HETEs in hObs, female -derived bone cells from both ages as well as male hObs produce both 12 and 15HETEs corrected for protein levels (12 H: 1.7+0.15 in pre-, 1.0+0.1 in post- and 0.2+0.05pg/ml in male hObs; 15H: 1.3+0.05 in pre-, 1.5+0.087 in post- and 0.22+0.03pg/ml in male hObs) (fig 1).

3.4. Modulation by JKF, F, D or E₂ of 12 and 15HETEs production in hObs

To investigate the hormonal responses of hObs, female-derived bone cells from both ages as well as male hObs produce both 12 and 15HETEs corrected for protein levels. Treatment with the different estrogenic hormones:E₂, D and F as well as JKF modulated the production of 12 and 15HETEs to different extents in both female age groups and only by JKF in male hObs (fig 3). These data suggest differences in the hormonal response of both LO products, namely 12 and 15HETEs.

Fig 3: The effects of treatment with E₂, D, F or JKF on 12 (upper panel) and 15 (lower panel) HETEs production in pre- and post- menopausal and male- hObs. Cells were incubated for 24 h with F (20µg/ml), D (300 nM) or E₂ (3 nM) or JKF (1 nM). Results are expressed as % change in the 12 and 15HETEs production (n=4–8). *P<0.05; **p<0.01, compared with control incubates containing the vehicle for the active compound only.

3.5 The modulation by JKF, F, D or E₂ of ROS formation in human hObs

To investigate the hormonal responses of hObs, female-derived bone cells (hObs) from pre- menopausal females produce reactive oxygen species (ROS) as analyzed histologically (fig 4) as well as chemically (fig 5). Treatment with the different estrogenic hormones as well as JKF modulated the production of ROS to different extents in both female age groups but not even by JKF in male hObs (fig 5). These data suggest differences in the hormonal response of ROS formation in an age and sex specific manner.

Fig 4: The effects of E₂, D, F or JKF on the production of ROS in cultured post- menopausal female measured by Histological analysis. Cells were incubated for 24 h with F (20µg/ml), D (30 nM) or E₂ (3 nM) or JKF (1 nM). Details are given in the experimental section.

Fig 5: The effects of treatment with E₂, D, F or JKF on ROS formation in pre- and post- menopausal and male hObs. Cells were incubated for 24 h with F (20µg/ml), D (30 nM) or E₂ (3 nM) or JKF (1 nM). Results are expressed as % change in ROS production (n=4–8). *P<0.05; **p<0.01, compared with control incubates containing the vehicle for the active compounds only.

3.6. The effect 12 or 15HETE on ROS formation in human hObs

To investigate the response of female-derived bone cells (hObs) from pre- menopausal females to LO products in terms of reactive oxygen species (ROS) production, cells were treated with12 and 15HETE at 1µM and then for 60 min with the fluorescent probe DFC and resulted in fluorescent labeling of the cells. Fluorescent microscopy of the cells showed staining with all compounds (fig 6). Fluorescent microscopy of the cells showed staining with all compounds which was blocked completely by pre-incubation for 30 min with DPI (data not shown). These data suggest that the LO products namely 12 and 15HETEs induce ROS formation in female- derived hObs.

Fig 6: The effects of 12 and 15HETEs (100pg/ml) on the production of ROS in cultured post- menopausal female measured by Histological analysis. Details are given in the experimental section.

4. Discussion

The effects of estrogenic compounds and vitamin D analogs on primary human female derived cultured hObs from both age groups; pre- and post-menopausal females as well as males complements our findings in human skeletal cell lines in vitro, where E₂ stimulate both cell proliferation and energy metabolism as well as membrane binding mediated responses (20-21). Although reports suggest that 12 and 15HETE interact with multiple signals promoting cell replication, their effects are probably not redundant, since inhibition of their production hinders normal cell growth in a variety of cell types (22). Recent publications linked also 12/15LO or 15LO and 12LO (platelet type) to bone density (23). In this study we provide a direct evidence for the expression and biological role of LOs in bone cell biology using primary cultures if human bone cells. We found mRNA expression of three types of LOs in cultured human bone cells and lines, i.e., the platelet type 12LO, 15LO type 1 and 15LO type 2 (11). We now show that the expression of the LOs in these cells is not only modulated by vitamin D metabolites (12), PTH (11), or E₂ (12,24), but also by phytoestrogens daidzein (D) and femarelle (F) and the vitamin D analog (JKF). The expression of these enzymes results in the ability of hObs to secrete 12HETE and 15HETE, the products of LOs. Moreover, the generation of HETE which is driven by the estrogenic compounds is linked to hObs growth induced by these hormones. The HETE raise local oxidative stress as was shown by us previously (12) by measuring ROS formation induced by estrogens in SaSO2 cells (6) and is complemented now in different hObs. Because oxidative stress, in turn, may lead to inhibition of differentiation of bone osteoblasts-like cells (23, 25,26) and acceleration of osteoclast differentiation (10), its induction may also result in the release of oxidizing fatty acids which unfavourably affect overall bone osteoblast / osteoclast homeostasis through enhanced oxidative stress. It is of interest to notice that the response of these parameters to gonadal steroids and phytoestrogenic products unlike other parameters connected to cell growth is sex-specific to certain extent.

Although reports suggest that 12 and 15HETE interact with multiple signals promoting cell replication, their effects are probably not redundant, since inhibition of their production hinders normal cell growth in a variety of cell types (25,26). Recent publications linked also 12/15LO or 15LO and 12LO (platelet type) to bone density (10,26).

In the present study we provide direct evidence for the expression and biological role of LO in bone cell biology. We found that cultured human bone cells express mRNA of three types of LO, i.e., the platelet type 12LO, 15LO type 1 and 15LO type 2 (2). Also the expression of the LO in these cells is modulated by a variety of hormones. The expression of these enzymes results in the ability of bone cells to form and secrete 12HETE and 15HETE, the products of LO. Moreover, the generation of 12/15HETE is linked to hormone-dependent osteoblastic growth. The 12/15HETE raise local oxidative stress as was shown here by measurements of ROS formation induced by the hormones as well as by the 12/15HETE themselves. Because oxidative stress, in turn, may lead to inhibition of differentiation of bone osteoblasts-like cells (25) and acceleration of osteoclast differentiation (26), its induction may also result in the release of oxidizing fatty acids which unfavourably affect overall bone osteoblast/ osteoclast homeostasis through enhanced oxidative stress. Presently, however, these potential secondary sequels of hormonal-stimulated LO activities in hObs remain entirely conjectural and are the subject of future investigation.

In conclusion, our data suggest that like E₂, the phytoestrogens and the vitamin D non-calcemic analog modulate hObs from pre- and post- menopausal females and in male hObs and the induction of lipoxygenase enzymes and their activity as well as ROS formation in an unclear mechanism (s), but still with sex-specificity. But the mechanism of F is different than E₂ and the other phytoestrogens which might leed to its problems effects which need further studies.

5. Materials and Methods

5.1. Cell cultures

Primary human bone cells (hObs) from pre- and post-menopausal females as well as males at different ages were prepared by us (27). Human bone cells were prepared from biopsies of patients undergoing corrective surgery following accidental injury, hip or knee replacement. All patients signed informed consent and were healthy, non-osteoporotic and not receiving hormonal replacement treatment. We studied both pre-menopausal women, whose ages ranged between 37–50 years (n = 5) and post-menopausal women, with an age range of 59–84 years (n = 5) and males between40–75 years (n = 7). The non-enzymatic method for isolation and culture of human bone cells and their characterization as osteoblasts were previously described us (1). Briefly, unused fragments of the trabecular surface of the iliac crest used for corrective spinal surgery were cut into 1 mm³ pieces and extensively and repeatedly washed with phosphate buffered saline (PBS) to remove blood components. The explants, with no enzymatic digestion, were seeded in 100 mm diameter tissue culture dishes and incubated in DMEM medium containing 10% fetal calf serum (FCS) and antibiotics, in the absence of Ca⁺⁺, to avoid fibroblastic, chondroblastic and osteoclastic growth (19,27). Cell outgrowth from the bone explants was apparent after 6–10 days. The culture cell population consisted of osteoblast-like cells (with a negligible number of fibroblasts) showing osteoblastic properties (1). First passage cells were seeded in phenol red free DMEM with 10% charcoal stripped FCS and incubated at 37°C in 5% CO₂.

5.2. Hormonal treatment

a. Sub-confluent cells were treated daily with vehicle, E₂ at 3nM, D at 300 nM or F at 20µg/ml or JKF at 1nM, for 3 days daily for the expression of mRNA for 12LO and 15LO type 1 expression were determined as previously described (11-12) or; b. for 1 hour with serum-free medium, followed by the addition of vehicle or the different hormones for 10 minutes and HETEs were extracted and assayed as previously described (11,12) or; c. for 1 hour with vehicle or estrogenic compounds or JKF and ROS was assayed as previously described (12).

5.3 Determination of mRNA for 12 and 15LO by RT-PCR

RNA was extracted and expression of 12 and 15LO enzymes was carried out by RT-PCR as described previously (11,12). RNA was extracted from cultured human bone cells as previously reported (11). Initial detection of the expression of 12- and 15-LO enzymes was carried out by RT-PCR as previously described (11). For subsequent quantification of the hormonal effect on 12-LO expression, amplification of the resulting cDNAwas performed in 25 µL on 96-well plates in a reactionbuffer containing Taqman universal PCR master mixture. RNAse P expression served as an internal control for each sampleand was measured by assay-on-demand gene expression products, which consist of a x 20 mixture of unlabeled PCR primers andTaqman MGB probe labeled with 5-carboxy fluorescein dye.

5.4 Determination of the levels of 12 and 15HETE by HPLC

Cells and medium were extracted for HETE and analyzed by HPLC as described previously (11). Protein was assayed by Coomassie brilliant blue dye binding, using BSA as the standard.

5.5. Determination of ROS formation

After hormonal treatment for 1 hour, and ROS formation using NBT colorimetric method as previously described (12) or for fluorescent microscopy by using 2′,7′-dichloro-fluorescein diacetate (DCF) was determined (12).

5.6 Statistical analysis

The significance of differences between experimental and control means was evaluated using Student's t-test, in which n = 5 number of cultures. p> 0.05, was considered significant. Values are means± S.E.M.