Mirdametinib

Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells

M. Zavatti a,⁎, M. Guida b, T. Maraldi a, F. Beretti a, L. Bertoni a, G.B. La Sala c, A. De Pol a

a b s t r a c t

Aims: Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance os- teoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteo- blastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.
Main methods: hAFSCs were cultured in a standard medium or in an osteoblastic medium for 14 or 21 days and ferutinin was added at 10−8 M. Immunofluorescence techniques and Western-blot 21analysis were used to study estrogen receptors and signaling pathways.
Key findings: In both undifferentiated and differentiated hAFSCs we identified ERα and GPR30 with a nuclear or cytoplasmatic localization, respectively. The presence of ferutinin in the osteoblastic medium leads to an increase in ERα expression. To dissect the role of estrogen receptors, MPP and G15 were used to selectively block ERα and GPR30, respectively. Notably, ferutinin enhanced osteoblastic differentiation in cells challenged with G15. Ferutinin was able to increase ERK and Akt phosphorylations with a different timing activation. These phosphor- ylations were antagonized by PD0325901, a MEK inhibitor, and wortmannin, a PI3K inhibitor. Both MPP and G15 inhibited the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. In the osteoblastic condition, PD0325901, but not wortmannin, reduced the expression of OPN and RUNX-2, whereas ferutinin abrogated the down-modulation triggered by PD0325901.
Significance: PI3K/Akt pathways seems to mediate the enhancement of hAFSCs osteoblastic differentiation trig- gered by ferutinin through ERα.

Keywords: Ferutinin hAFSCs Osteoblastic differentiation Estrogen receptor signaling

1. Introduction

Ferutinin (jaeschkeanadiol p-hydroxybenzoate) is a daucane phyto- estrogen found in the plants of Ferula genus (Umbelliferae). It has been demonstrated that ferutinin has an estrogenic activity because it is able to bind estrogen receptors [1,2]. In vitro experiments showed a higher affinity for estrogen receptor (ER) α (IC50 = 33.1 nM) than for ERβ (IC50 = 180.5 nM) [2]. Moreover, ferutinin has an important role in bone metabolism since it is able to prevent and to treat osteoporosis in- duced by estrogen deficiency in ovariectomized rats [3]. The effects on bone mass exerted by ferutinin are comparable to those exerted by es- tradiol benzoate [4,5]. Recently, we demonstrated that ferutinin, through ERα, enhances bone reconstruction, when orally administered in rats with a calvarias critical size bone defect, filled with a collagen type 1 and human amniotic fluid stem cells (hAFSCs) construct [6]. This construct leads to an approximately 70% bone reconstruction showing that ferutinin could act, as a healing promoting factor, on hAFSCs inducing osteogenic differentiation [6,7]. Among stem cells, hAFSCs attracted the interest of researchers because they are placed midway between embryonic and adult stem cells, are easily to obtain without ethical problems and can be maintained in culture without dif- ficulties [8,9]. hAFSCs are able to differentiate toward osteogenic lineage under suitable conditions such as the presence of ascorbic acid that stimulates extracellular matrix (ECM) synthesis, dexamethasone that stimulates cellular differentiation and β-glycerophosphate that pro- motes ECM mineralization [10]. In our laboratory, the osteogenic poten- tial of hAFSCs was also demonstrated in vitro and in vivo on three- dimensional surface of poly-D,L-lactic acid, fibroin, or collagen [11,12]. Regarding the effects of ferutinin
(MAPK) signaling pathways are involved in cell survival, proliferation and differentiation [13,14] and in particular ERK signaling pathway stimulates osteogenic differentiation of stem cells [15]. Many phytoestrogens exerted their stimulating effects on osteoblastic differ- entiation via ERK signaling. Resveratrol stimulates human bone mar- row-derived mesenchymal stem cells (BMSCs) proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation [16]. Xanthoumol stimulates osteoblastic differentiation by activation of RUNX-2 via a mechanism related to the p38 MAPK and ERK signaling pathways in MC3T3-E1 and C2C12 cells [17]. Quercetin promotes proliferation, osteogenic differentiation and angiogenic factor secretion of BMSCs through ERK and p38 signaling pathways [18]. Also the PI3-kinase-Akt pathways [19,20] is an impor- tant player of the osteogenic network as demonstrated by Mukherjee and Rotwein [21]. They showed that Akt activity is required for all stages of osteoblast differentiation (lineage commitment, early differentiation and maturation). Among phytoestrogens, icariin exerts its osteogenic effect on rat BMSCs activating the PI3K-Akt signaling pathways [22]. Puerarin, an isoflavone glycoside from Pueraria lobate (Willd.), stimu- lates bone formation through activation of PI3K/Akt pathways in rat calvarial osteoblasts [23].
The current study was carried out to explain a possible molecular mechanism of ferutinin-induced osteoblastic differentiation of hAFSCs, through ERα and GPR30, evaluating the role of the MEK/ERK and PI3K/Akt signaling pathways.

2. Materials and methods

2.1. Cell culture and treatments

Supernumerary amniocentesis samples were provided by the Laboratorio di Citogenetica, Arcispedale Santa Maria Nuova (Reggio Emilia, Italy). All samples were collected with informed consent of pa- tients according to Italian law and ethical committee guidelines (Proto- col n° 2015/0,004,362 of 02/24/2015). Amniotic fluid stem cells (AFSCs) were isolated according to De Coppi et al. [8] and Maraldi et al. [11]. Briefly, human amniocentesis cultures were harvested by trypsinization and subjected to c-Kit immunoselection by MACS® technology (Miltenyi Biotec, Cologne, Germany) [11]. c-Kit positive cells were subcultured routinely at 1:6 dilution and not allowed to expand beyond a 80% of confluence. AFSCs were grown in a minimum essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all reagents, EuroClone, Milan, Italy) at 37 °C and 5% CO2. Ferutinin (Indena SpA, Milan, Italy) was solubilized in dimethyl sulfoxide (DMSO) and added to the culture medium at the concentration of 10−8 M.
In the ERK and Akt pathways studies ferutinin was added to cell me- dium for different time periods (5′, 15′, 30′, 1 h or 2 h) whereas the MEK inhibitor PD0325901 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 10 μM and the PI3Kinase inhibitor wortmannin (Santa Cruz Biotechnol- ogy, Santa Cruz, CA, USA) at 100 nM were added 1 h prior to ferutinin. The ERα antagonist methyl-piperidino-pyrazole hydrate (MPP) (Sigma Aldrich, St Louis, MO, USA) and the GPR30 antagonist 4-(6- Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15) (Tocris Bioscience, Bristol, UK) were used at the concentration of 1 μM, 1 h prior to ferutinin.

2.2. Osteoblastic differentiation

Cells were seeded approximately 3000 cells/cm2 on culture dishes. When over 80% of confluence was reached, the standard medium was replaced with the osteoblastic one consisting of αMEM with 10% FBS, 100 μM 2P-ascorbic acid, 100 nM dexamethasone, 10 mM β- glyrocerophosphate (all from Sigma-Aldrich, St Louis, MO, USA), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin [24]. The culture medium was refreshed every 3–4 days until 21 days of culture. In the osteogenic conditions ferutinin was used at the con- centration of 10−8 M, MPP and G15 at 1 μM. To analyze signaling pathways during osteoblastic differentiation, AFSCs were pretreated with PD0325901 10 μM or wortmannin 100 nM for 1 h and then incubated with ferutinin for 14 days.

2.3. Western blot analysis

Whole cell lysates obtained from AFSCs at two different conditions of culture (osteogenic or not) were harvested, washed with phosphate buffered saline (PBS), and gently lysed on ice for 10 min in hypotonic lysis buffer (20 mM Tris-HCl, pH 7, containing 1% Nonidet P40, 150 mM NaCl, 10% Glycerol, 10 mM EDTA, 20 mM NaF, 5 mM Sodium Pyrophosphate, 1 mM Na3VO4, and freshly added Sigma-Aldrich Prote- ase Inhibitor Cocktail). After sonication, lysates were cleared by centri- fugation for 15 min at 14,000 g in a refrigerated centrifuge. The supernatants were collected and the protein concentrations were deter- mined by Bradford assay. 60 μg of protein for each sample were separat- ed by 8 or 10% SDS page and then transferred to PVDF membrane. The Western blot technique was performed as described previously [25]. Briefly, the membranes were blocked with 3% dry milk and 2% bovine serum albumin (BSA) in Tris-buffered saline–Tween 20 (0.1%) (TBS-T) for 30 min. Blots were incubated overnight at 4 °C with one of the fol- lowing primary antibodies (Abs), diluted 1:700 in TBS-T + 2% BSA and 3% milk: mouse anti-ERα (Millipore, Billerica, MA, USA), rabbit anti-GPR30 (Genetex, Irvine, CA, USA), rabbit anti pERK (DB Biotech, Ko- sice, Slovakia, EU), rabbit anti-pAKT(s473) and rabbit anti-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Runx2 and mouse anti-Osteopontin (Abcam, Cambridge, UK), goat anti-Akt1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing three times in blocking buffer, membranes were incubated with peroxidase-labelled anti-rabbit, anti-mouse or anti-goat secondary Abs diluted 1:3000, for 1 h at room temperature. All membranes were visualized using ECL (en- hanced chemioluminescence, Amersham, UK). Goat anti-βactin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-ERK1/2 or goat anti-Akt1/2 were used as protein loading control. Densitometry was performed on three independent experiments by ImageJ Data Analyzer software. The relative expression of each protein was quantified and normalized to the corresponding protein loading control.

2.4. Immunofluorescence and confocal microscopy

Monolayer cells were fixed in 4% paraformaldehyde in PBS at pH 7.4 for 20 min and then processed for subsequent immunofluorescence or histological staining.
Fixed monolayer cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min to detect nuclear and cytosolic markers. Permeabilized and not permeabilized samples were washed three times with PBS and then blocked with 3% BSA in PBS for 30 min at RT. Samples were in- cubated with the primary antibodies diluted 1:50 in PBS containing 3% BSA: mouse anti-ERα and rabbit anti-Osteocalcin (OCN) (Millipore, Bil- lerica, MA, USA), rabbit anti-GPR30 (Genetex, Irvine, CA, USA), mouse anti-OPN (Abcam, Cambridge, UK), rabbit anti-Runx2, for 1 h at RT.
After washing with PBS 3% BSA, the samples were incubated for 1 h at RT with the secondary antibody diluted 1:100 in PBS 3% BSA: goat anti-mouse Alexa fluor® 488, donkey anti-rabbit-Alexa fluor® 647 (Life Technologies, Carlsbad, CA, USA). After washing in PBS, samples were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) in H2O for 5 min and then mounted with anti-fading medium, 0.21 M DABCO (1,4-diazabicyclo[2.2.2]octane) and 90% glycerol in 0.02 M Tris, pH 8.0. Negative controls consisted of samples not incubated with the primary antibody.
The multi-labeling immunofluorescence experiments were carried out avoiding cross-reactions between primary and secondary antibodies. Confocal imaging was performed by a Nikon A1 confocal laser-scan- ning microscope. Spectral analysis was performed to exclude overlap- ping between two signals or the influence of autofluorescence on the fluorochrome signals. The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections. The image rendering was performed by Adobe Photoshop Software (Adobe System Software, Ireland).

2.5. Alizarin Red S staining and mineralization

Fixed monolayer cells were washed with distilled water and then in- cubated for 10 min at room temperature in a solution containing 2% of Alizarin Red S at pH 4.2. Images of histological samples were obtained with a Zeiss Axiophot microscope (Zeiss AG, Jena, Germany), equipped with a Nikon DS-5Mc CCD colour camera.
To quantify the Alizarin Red S staining, samples were washed three times with PBS and then 1 mL of 10% cetylpyridinium chloride was added to each well and incubated for 20 min to elute the stain. 100 μL of this eluted stain were added to 96 well plates and read at 485 nm using a spectrophometer (Appliskan, Thermo Scientific, Finland) [26].

2.6. Statistical analysis

Statistical analysis was assessed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). All data were expressed as mean ± SEM. One way-analysis of variance (ANOVA) with Newman-Keuls post-test or Dunnett’s Multiple Comparison test was used. P b 0.05 was taken as the level of significance.

3. Results

3.1. Ferutinin-promoted hAFSCs osteoblastic differentiation is mediated through estrogen receptor ERα

Based on our previous work demonstrating that ferutinin enhances osteoblastic differentiation of hAFSCs, we asked whether this action is mediated through known estrogen receptors. Thus, cells were grown in the presence of the osteoblastic medium for 21 days with or without ferutinin. As expected, ferutinin is indeed sufficient to increase calcium deposition and mineralization, as shown by Alizarin Red S staining of calcium nodules (Fig. 1 A). We observed a statistically significant in- crease of calcium deposition in the hAFSCs treated with the osteoblastic medium (P b 0.01) with or without ferutinin supplement, as compared with a standard medium (Fig. 1 B). Moreover, the treatment with ferutinin led to a significant (P b 0.05) higher value of absorbance in comparison with the osteoblastic control.
Thus, we next assessed the presence and localization of ERs by immunofluorescence with antibodies to ERα and GPR30. Fig. 2 (A–D) shows that both receptors are expressed in undifferentiated (Fig. 2 A–B) as well as in osteoblastic differentiated cells (Fig. 2 C– D), although with a different subcellular distribution: ERα localized to the nucleus (Fig. 2 A–C), whereas GPR30 was restricted to the cytoplasm (Fig. 2 B–D). Moreover, while ERα level increased in oste- oblastic differentiated cells, GPR30 level did not undergo any change. This result was confirmed by Western blot, where densitometric analysis showed that ERα was significantly (P b 0.05) higher in the osteoblastic differentiation and was further increased upon addition of ferutinin to the osteoblastic medium for 21 days (P b 0.01) (Fig. 2 E–F). As expected, a ferutinin-dependent boost of the expression of the os- teogenic markers osteopontin (OPN) and osteocalcin (OCN) was ob- served (Fig. 3). Next, cells were treated with ERα antagonist MPP and GPR30 antagonist G15, in the presence or not of ferutinin, followed by tri- ple immunofluorescence labeling to stain nuclei and localize OPN and OCN (Fig. 3). Notably, blocking GPR30 led to enhanced osteoblastic differ- entiation markers, an event further increased by co-addition of ferutinin. Conversely, the ERα antagonist MPP prevented osteoblastic differentia- tion. Co-addition of ferutinin partially recovered OPN, but not OCN, ex- pression. We concluded that osteoblastic differentiation in this cell model depends on fine balancing of the activity of these two receptors, and that ferutinin pro-differentiation function is mediated, at least in part, through ERα.

3.2. Effects of ferutinin on p-ERK and p-Akt in hAFSCs. Involvement of MEK/ ERK and PI3K/Akt pathways on osteoblastic differentiation of hAFSCs

Because ERα and GPR30 are known to activate a plethora of second messengers in response to 17β-estradiol, we wondered whether ferutinin is able to trigger signaling likewise. Western-blot analysis, with antibodies recognizing epitopes directly related to their kinase ac- tivity, revealed that ferutinin activates both ERK and Akt as soon as 5 min after treatment of hAFSCs, although with a different timing (Fig. 4 A). In particular, p-ERK peaked 15 min after ferutinin addition and remained constant for up to 1 h (Fig. 4 B), whereas Akt activation peaked at 5 min, then returned to basal level. The effect of inhibition of the two pathways on ferutinin action was therefore investigated by addition to hAFSCs medium of the specific drugs PD0325901 and wortmannin. As expected, p-ERK and p-Akt levels were significantly re- duced by both PD0325901 and wortmannin (Fig. 4 C–F). Remarkably, however, the effects of both drugs were counteracted by ferutinin (P b 0.01), leading to a partial recovery of ERK activity and to a complete recovery of Akt activity.
Next, the effect of ER antagonists on the above pathways was an- alyzed. Cells were incubated for 1 h with G15 or MPP prior to addi- tion of ferutinin to the culture medium. While neither of them displayed any effect if added alone, it is worth noting that both drugs significantly prevented ERK and Akt activation triggered by ferutinin, although to a different extent (P b 0.0001 for pERK and P b 0.05 for pAkt) (Fig. 5 A–B).
Then, the effect of blocking MEK/ERK and PI3K/Akt signaling on ferutinin-induced accumulation of osteoblastic markers was inves- tigated by means of PD0325901 or wortmannin. It is important to note that, besides the expected accumulation of OPN and the in- crease of RUNX-2 (Fig. 6 A), ferutinin was able to partially recover the drop of OPN and RUNX-2 levels triggered by PD0325901. The densitometric analysis on Western-blot bands showed a significant increase of RUNX-2 (P b 0.0001) and OPN (P b 0.01) levels in ferutinin-treated cells in comparison to control group (Fig. 6 B). On the contrary, wortmannin displayed no effects on either RUNX-2 or OPN.

4. Discussion

In this study we sought to explain the mechanism of action underly- ing ferutinin stimulation of osteoblastic differentiation of hAFSCs. In our previous work we reported the ability of ferutinin to enhance osteoblas- tic differentiation of hAFSCs in vitro [7] as well as that to improve regen- eration of critical-size bone defects in vivo, when administered to rats treated with a collagen-hAFSCs construct, and we hypothesized that ferutinin acted through binding to the classical estrogen receptor ERα [6]. Thus, we explored the involvement both of ERα and of the mem- brane-associated receptor GPR30 in ferutinin-stimulated osteoblastic differentiation in vitro. Both ERs are known to be expressed in all human bone cell types, namely osteoblast, osteocyte and osteoclast [27–30]. Our results showed that ERα and GPR30 are expressed in both undifferentiated and differentiated hAFSC, although ERα is re- stricted to the nucleus and is expressed at higher amount in differenti- ated cells, while GPR30 shows a cytoplasmic localization and its amount does not differ among between the two conditions. The in- creased level of ERα during osteoblastic differentiation is in keeping with what previously observed in rat calvarial osteoblasts [31]. More- over, also the observation that the presence of ferutinin in the osteoblas- tic differentiating medium leads to a further significant increase in ERα expression, is in agreement with our previous in vivo results, showing that ferutinin treatment up-regulates the amount of ERα in the bone and in the connective tissue [6]. Interestingly, these actions of ferutinin are reminiscent of the effects recently described for the phytoestrogen puerarin (daidzein-8-C-glucoside), that also enhances osteoblastic dif- ferentiation primarily through ERα [32]. Furthermore, we evaluated the involvement of ERα and GPR30 in the ferutinin-induced osteoblas- tic differentiation of hAFSCs, also by challenging these receptors with their specific antagonists MPP and G15. After 21 days, in the presence of the osteoblastic medium, the osteogenic markers OPN and OCN were highly expressed in the presence of ferutinin, as expected, and in- hibition of GPR30 by G15, both alone and with ferutinin, evokes an ad- ditional boost in the level of both markers, in comparison to the osteogenic control. We concluded that G15 potentiates ferutinin effect by restricting ferutinin binding to ERα only. Conversely, when ERα was antagonized by MPP, osteoblastic differentiation was markedly re- duced, whereas the presence of ferutinin partially counteracted MPP ef- fects. This is particularly true in the case of OPN. Because OPN is expressed from the early stages of osteoblastic differentiation, whereas OCN is expressed only post-proliferatively with the onset of nodule for- mation [33,34], this differential temporal expression of OPN and OCN could explain why in the MPP + F condition we observed an increase only in the OPN level.
Furthermore, we addressed the issue of the signaling pathways re- sponsible for ferutinin-induced osteoblastic differentiation in hAFSCs. We decided to investigate MEK/ERK and PI3K/Akt signaling pathways since they are involved in the osteoblastic differentiation [35]. Ferutinin activated these pathways starting from 5 min after exposure. In partic- ular, the phosphorylation of ERK reached a peak after 15 min and the ef- fect lasted for 1 h after exposure, whereas Akt phosphorylation peaked 5 min after exposure and rapidly returned to basal levels. These phos- phorylations were antagonized by PD0325901, a MEK specific inhibitor, and wortmannin, a PI3K specific inhibitor. Activation of the above key signaling molecules may be accounted for by binding of ferutinin to classical or non-classical estrogen receptors, such as steroid hormone receptors localized at the plasma membrane, or to the non-steroid hor- mone receptor GPR30. Both mechanisms were characterized by previ- ous reports demonstrating that MEK/ERK and PI3K/Akt activations by 17β-estradiol were mediated via both classical ERs and GPR30 receptors [36,37]. Estrogens acted through classical nuclear ERs on gene expres- sion exerting their genomic effects. In addition to the long-term (from hours to days) regulation of gene expression, estrogens also mediates rapid (from seconds to minutes) signaling events (non-genomic effects) such as the activation of cellular kinases [38]. However, there was not a clear distinction between genomic and non-genomic effects because many intracellular pathways lead to a modulation of gene expression [39].
To evaluate which pathway was involved in our system, we treated undifferentiated cells with the antagonists MPP for ERα or G15 for GPR30 for 30 min prior to ferutinin application. MPP and G15 behaved the same way inhibiting the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. Since both ERα and GPR30 are expressed in hAFSCs, an explanation for this result might be the cross-activation of their downstream signaling. Indeed, functional cross-talk between ERα and GPR30 has been already reported in vascular studies and in es- trogen-mediated activity in cancer cells [40–42].
Next, we explored the role of MEK/ERK and PI3K/Akt pathways in the ferutinin-enhancing osteoblastic differentiation, a long term condi- tion in which we already know that the markers of the early stages of osteoblastic differentiation OPN and RUNX-2 are markedly expressed in hAFSCs [7,33]. Cells were pre-treated with PD0325901 or wortmannin for 1 h and then incubated with ferutinin in an osteogenic medium, for 14 days, then expression of OPN and RUNX-2 was moni- tored. Here PD0325901, but not wortmannin, efficiently reduced the ex- pression of both proteins, whereas ferutinin not only significantly increased RUNX-2 and OPN levels when added alone, as expected, but was also able to abrogate the down-modulation triggered by PD0325901, leading to a significant increase in the osteoblastic markers in comparison to PD0325901 alone. This indicates that signaling by MEK/ERK modulates osteoblastic differentiation. Conversely, the PI3K/ Akt pathways seems to mediate the enhancement of hAFSCs osteoblas- tic differentiation triggered by ferutinin through ERα.
Further studies are needed to define the role of ERs in the ferutinin- induced osteoblastic differentiation throughout the interactions be- tween classical and non classical ERs in genomic and non-genomic responses.

5. Conclusion

In conclusion, our study indicates that ferutinin is able to stimulate both MEK/ERK and PI3K/Akt signaling in undifferentiated hAFSCs, al- though with different timing of the phosphorylation pattern. Moreover, in the canonical osteoblastic differentiation model, both pathways were involved, but PI3K/Akt is required to ferutinin stimulated osteoblastic dif- ferentiation through ERα. The role of GPR30 should be clarified taking into account the interactions occurring between GPR30 and classical ERs pathways. Overall, these results strengthen the concept of ferutinin as an interesting phytoestrogens with an osteoinductive potential that might be applicable to the treatment of osteoporosis or in the bone tissue engineering.

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