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. 2016 Jul;231(7):1586-92.
doi: 10.1002/jcp.25254. Epub 2015 Nov 26.

Sex Steroids Influence Brain-Derived Neurotropic Factor Secretion From Human Airway Smooth Muscle Cells

Sheng-Yu Wang  1   2 , Michelle R Freeman  2 , Venkatachalem Sathish  2   3 , Michael A Thompson  2 , Christina M Pabelick  2   3 , Y S Prakash  2   3
Affiliations

Sex Steroids Influence Brain-Derived Neurotropic Factor Secretion From Human Airway Smooth Muscle Cells

Sheng-Yu Wang et al. J Cell Physiol. 2016 Jul.

Abstract

Brain derived neurotropic factor (BDNF) is emerging as an important player in airway inflammation, remodeling, and hyperreactivity. Separately, there is increasing evidence that sex hormones contribute to pathophysiology in the lung. BDNF and sex steroid signaling are thought to be intricately linked in the brain. There is currently little information on BDNF and sex steroid interactions in the airway but is relevant to understanding growth factor signaling in the context of asthma in men versus women. In this study, we assessed the effect of sex steroids on BDNF expression and secretion in human airway smooth muscle (ASM). Human ASM was treated with estrogen (E2 ) or testosterone (T, 10 nM each) and intracellular BDNF and secreted BDNF measured. E2 and T significantly reduced secretion of BDNF; effects prevented by estrogen and androgen receptor inhibitor, ICI 182,780 (1 μM), and flutamide (10 μM), respectively. Interestingly, no significant changes were observed in intracellular BDNF mRNA or protein expression. High affinity BDNF receptor, TrkB, was not altered by E2 or T. E2 (but not T) significantly increased intracellular cyclic AMP levels. Notably, Epac1 and Epac2 expression were significantly reduced by E2 and T. Furthermore, SNARE complex protein SNAP25 was decreased. Overall, these novel data suggest that physiologically relevant concentrations of E2 or T inhibit BDNF secretion in human ASM, suggesting a potential interaction of sex steroids with BDNF in the airway that is different from brain. The relevance of sex steroid-BDNF interactions may lie in their overall contribution to airway diseases such as asthma.

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Figures

Figure 1
Figure 1. Effect of estrogen (E2) and Testosterone (T) on secretion of brain-derived neurotrophic factor (BDNF) in human airway smooth muscle (ASM)
A) BDNF secretion by human ASM cells was significantly decreased following E2 or T treatment. B) Similarly, 48h exposure of E2 or T significantly reduced BDNF secretion from fresh human ASM tissue. C) BDNF release was significantly decreased in tissue treated conditioned media. Released BNDF was normalized to GAPDH from matching tissue homogenate. Data are presented as mean ± SE, n = minimum of 4 patients, p < 0.05. *indicates significant effect of E2 or T from vehicle.
Figure 2
Figure 2. Effect of E2 and T on BDNF expression in human ASM
A) Exposure of E2 or T (24 h) did not alter BDNF mRNA expression in human ASM cells (measured by qPCR). B) No significant difference in BDNF expression was observed via western blot analysis of cell lysates following exposure to E2 or T (48 h). Bar graph is total pro-BDNF and BDNF expression normalized to β-actin. Data are presented as mean ± SE, no = minimum of 4 patients, p < 0.05. *indicates significant effect of E2 or T from vehicle.
Figure 3
Figure 3. Effect of E2 and T on TrkB expression in human ASM
A) Western blot analysis of TrkB-FL and TrkB-T1 in human ASM cells exposed to E2 or T for 48h. B) There was no significant change in TrkB-FL expression between experimental groups, C) Conversely, both E2 and T treatment significantly increased TrkB-T1 expression. D) However, the ratio of TrkB-FL to TrkB-T1 was found not to be significantly different. Data are presented as mean ± SE, n = minimum of 4 patients, p < 0.05. *indicates significant effect of E2 or T from vehicle.
Figure 4
Figure 4. Effect of estrogen or androgen receptor inhibition on BDNF secretion in human ASM
Human ASM cells were treated for 30 min with 1 μM estrogen receptor (ER) antagonist ICI 182,780 (ICI) or androgen receptor (AR) antagonist 10 μM Flutamide (FLU) prior to 48h exposure of E2 or T, respectively. A) BDNF secretion was restored to vehicle control in the presence of ICI and FLU. B) Western blot analysis of human ASM tissue conditioned media showed the same trend, but was not statistically significant. GAPDH from matching tissue homogenate was used to normalize the conditioned media. The experimental data are presented as mean ± SE, n = minimum of 4 patients, p < 0.05. *indicates significant effect of E2 and T from vehicle. #indicates significant effect of inhibitors from E2 or T.
Figure 5
Figure 5. Effect of E2 and T on cAMP levels and Epac expression in human ASM
A) Exposure of E2 significantly increased intracellular cAMP compared to vehicle. There was no significant effect with T exposure. B and C) Expression of the regulatory proteins Epac1 and Epac2 was significantly reduced in the presence of E2 or T. Data are presented as mean ± SE, n = minimum of 5 patients, p < 0.05. *indicates significant effect of E2 and T from vehicle.
Figure 6
Figure 6. Effect of E2 and T on secretory vesicular proteins (SNAP25 and SNAP 47) in human ASM
A) Exposure of E2 or T significantly decreased SNAP25 expression compared to vehicle. B) There was no significant difference in SNAP47 expression between experimental groups. Data are presented as mean ± SE, n = minimum of 5 patients, p < 0.05. *indicates significant effect of E2 and T from vehicle.
Figure 7
Figure 7. Potential mechanisms by which E2 and T affect BDNF secretion in human ASM
Inhibition of BDNF secretion (but not BDNF expression) in human ASM by sex steroids may be mediated via cAMP-Epac pathway and/or reduced SNAP25 vesicular protein expression.
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