The relationship between estrogen and the
The relationship between estrogen and the Hedgehog pathway as well as the associated mechanism have already been investigated in many tissues, especially in breast cancer (Chang et al., 2012, Katayama et al., 2006, Ramaswamy et al., 2012). Hitherto, even though we have proved that HH-Gli pathway promoted osteoclastogenesis in the condition of estrogen deficiency, the molecule which linked estrogen and the HH-Gli signaling pathway still needed to be investigated. As reported by some previous studies, in some tissues, MEK/ERK signaling was associated with Hedgehog signaling pathway. In human colon carcinoma, Gli1 transcription is inhibited when blocking ERK signaling with U0126 (Mazumdar et al., 2011), and the GLI1 level was decreased by the MEK1/2 inhibitor PD98059 in T-ALL cell lines (Hou et al., 2014). Although previous studies reported the promotion of the MEK/ERK pathway had positive effect on HH-Gli signaling, recent studies reported the opposite effect in specific tissues. Yang et al. detected a negative relationship between MEK/ERK signaling and the HH-Gli pathway in chondroid BAY85-3934 (Yang et al., 2013). Lauth et al. demonstrated that RAS signaling antagonizes autocrine HH signal transduction (Lauth et al., 2010). As for the relationship between estrogen and MEK/ERK signaling, many studies revealed positive or negative connection patterns between them in different tissues. Xie et al. suggested that estrogen promoted the activation of MEK/ERK signaling in mature osteoclasts (Xie et al., 2011). This highlighted the possible effect of MEK/ERK signaling in mediating estrogen and HH-Gli pathway in osteoclast. By our results, which suggested that MEK/ERK signaling was activated by estrogen and that the suppressed HH-Gli signaling in the condition of estrogen administration was upregulated when the MEK/ERK pathway was blocked by U0126 in osteoclasts, MEK/ERK signaling linked estrogen and HH-Gli signaling and mediated the suppressive effect of estrogen on HH-Gli signaling in osteoclast. Moreover, MEK/ERK signaling had a negative effect on osteoclast proliferation and differentiation and a positive effect on osteoclast apoptosis. These results further addressed the participation of MEK/ERK signaling in regulating the HH-Gli pathway in postmenopausal osteoporosis. Even though we proved that ERK signaling was involved in the relationship of estrogen and Hedgehog signaling and that estrogen positively regulated ERK signaling in osteoclasts, the mechanism underlying in the regulation of ERK signaling still remained unclear. Xie et al. suggested that estrogen receptor mediated different physical effects via ERK signaling of different activation period (Xie et al., 2011). A complex cascade which involved G protein-coupled estrogen receptor-1 and protein kinase C was proved to be related to the activation of ERK signaling regulated by estrogen (Grassi et al., 2015, Kabir et al., 2015). Therefore, the molecules involved in the activation cascade and the activation period of ERK signaling may be focused in further studies of the mechanism of the regulation of ERK signaling in osteoclasts. Sonic Hedgehog (SHH), which is an important Hedgehog ligand in maintaining adult bone homeostasis (James et al., 2012), was promoted in mRNA expression after we treated osteoclasts with U0126. We also detected an increase in the SHH levels in the cytoplasm of osteoclasts. Additionally, Shh had higher expression levels in cells treated with U0126 and 17β-estradiol compared with those treated with 17β-estradiol alone. These findings indicated that Shh expression was inhibited by MEK/ERK signaling and that SHH was the molecule linking estrogen with the HH-Gli pathway. When SHH was neutralized by an anti-SHH antibody, the Gli1 expression level was decreased in cells treated with U0126. Therefore, SHH acted as a bridge linking MEK/ERK signaling and the HH-Gli pathway in osteoclasts. In summary, estrogen activated MEK/ERK signaling, which inhibited the HH-Gli signaling pathway in osteoclasts. HH-Gli signaling promoted osteoclast proliferation and differentiation and inhibited osteoclast apoptosis. Therefore, estrogen inhibited osteoclast proliferation and differentiation and induced osteoclast apoptosis by inhibiting HH-Gli signaling. In postmenopausal osteoporosis, estrogen deficiency disturbed the activity of MEK/ERK signaling and unrepressed HH-Gli signaling in osteoclasts. Then, the upregulated HH-Gli pathway increased the numbers of osteoclasts. We elucidated the connection between estrogen and the Hedgehog signaling pathway as well as the underlying mechanism. However, further studies (especially in vivo experiments) are needed to validate these findings.