Similar to previous studies (7
), the current study clearly showed the existence of amiloride-sensitive epithelial sodium channels (ENaC) in skeletal cells, such as in chondrocytes and osteoblasts. We also observed that the mRNA and the protein level of ENaC α, γ in E2 treatment groups are higher than those in the control groups in the primary cultured osteoblasts. These results indicated that estrogen regulates the expression of ENaC α, γ in cultured osteoblasts. Furthermore, we found that estrogen increased the ENaC activity of osteoblasts, which was represented by the whole-cell amiloride-sensitive currents as observed by using the patch clamp method. The inner amiloride-sensitive currents implied that the inward Na+ currents and the perfusion of the bath solution containing 20 nM E2 greatly increased the recorded total currents, indicating the enhanced effects of E2 on the currents and that its stimulation on osteoblasts is rapid and extremely sensitive. This similar phenomenon also occurs in lung and kidney tissues.
One previous study (21
) found that female ovarian hormones up-regulate ENaC α, γ mRNA expression in lung tissue of female rats. Another study (10
) found that mRNA levels of ENaC subunits in the kidney were higher in female rats than in male rats; ovariectomy made no differences between the sexes. Moreover, the ovariectomized rats showed that estrogen up-regulates ENaC α mRNA levels in the kidney. The current study is consistent with these previous findings which reveal that estrogen also regulates ENaCs in primary cultured osteoblasts. However, ENaCs are tissue-specific and species- specific in their responses to estrogen. ENaC γ was increased by estrogen stimulation in rat lung tissue and mouse osteoblasts, and not in rat kidney tissue. The expression of β-ENaC In our experiments was not determined may related to these reason, the exact reason need to be explored in future.
Lastly, in our study, the expression of α, γ ENaC, mRNA and protein were increased when conditional medium promoted primary osteoblast differentiation, which indicated that the expressions of ENaCs varied at different stages of their development within the culture, meaning that ENaCs increase during osteoblast maturation. Our result shows the difference of expression between protein and mRNA level. There might be some reasons for it: 1. Protein can not only be regulated on transcription level but also translation level and translocation level. That is why mRNA is not a direct indication of protein level. 2. There is also one possibility though comparatively rare, the antibody epitope may be changed by alternative mRNA splicing.
Based on our findings, estrogen may serve as a new regulatory pathway through which ENaC can regulate osteoblast activity. Estrogen’s function of protection against the progression of bone disease has been well known. For example, estrogen deficiency in women can cause severe and rapid bone loss, which can be prevented or reversed by estrogen replacement. Moreover, the loss of estrogen activity was a leading cause for osteoporosis in men as well (2
Classical receptors for estrogens (ERα and ERβ) are present (4
) in bone, osteoblasts, osteoclasts and their progenitors, indicating that the effects of sex steroids on bone are at least, in part, directly mediated. However, the level of receptor expression in bone cells is low (at least 10-fold) compared to that in reproductive organs. Moreover, the level of receptor expression in bone cells does not vary by gender, whereas similar levels (1
) of ER have been found in both sexes. In addition to traditional effects, estrogen has traditionally been known for its indirect effects on systemic hormones, which regulate calcium balance, a change of the ratio among RANKL, cytokine-Inducing osteoclastogenesis, and osteoprotegerin (OPG), its decoy-soluble receptor (25
The findings of our study provide a new perspective on the regulatory roles of estrogen in ENaC expression and the functions of osteoblasts. Since half of the sodium in the body exits in bone (18
), we speculate that the elevated level of ENaCs may be due to estrogen increasing the amount of sodium entering into the osteoblasts, which may further influence bone formation and mineralization. Combining the above results, we postulate that ENaC may be involved in bone metabolism. More research needs to be done to further explore this area.
In summary, our study has confirmed the presence of ENaCs in osteoblasts, and has found that an increase in ENaC expression and function induced by estrogen in osteoblasts may relate to bone metabolism. The insight of the current study suggests that ENaCs may be a potential target for developing pharmaceutical intervention for metabolic bone diseases.