Sarmah Swapnalee, Curtis Courtney, Mahin Jennifer, Farrell Mark, Engler Thomas A, Sanchez-Felix Manuel V, Sato Masahiko, Ma Yanfai Linda, Chu Shaoyou, Marrs James A
Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
Lilly Research Laboratories, Indianapolis, IN 46225, USA.
Biomedicines. 2019 Apr 19;7(2):30. doi: 10.3390/biomedicines7020030.
The Wnt pathway has been shown to regulate bone homeostasis and to influence some bone disease states. We utilized a zebrafish model system to study the effects of a synthetic, orally bioavailable glycogen synthase kinase-3β (GSK3β) inhibitor LSN 2105786, which activates Wnt signaling during bone healing and embryogenesis. GSK3β inhibitor treatment was used to phenocopy GSK3β morpholino oligonucleotide (MO) knockdown in zebrafish embryos. Human and zebrafish synthetic mRNA injection were similarly effective at rescue of GSK3β MO knockdown. During caudal fin regeneration, bony rays are the first structure to differentiate in zebrafish fins, providing a useful model to study bone healing. Caudal fin regeneration experiments were conducted using various concentrations of a GSK3β inhibitor, examining duration and concentration dependence on regenerative outgrowth. Experiments revealed continuous low concentration (4-5 nM) treatment to be more effective at increasing regeneration than intermittent dosing. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. Increased Wnt responsive gene expression and differentiation were observed in response to GSK3b inhibitor treatment. Activating Wnt signaling also increased cell proliferation and osteoblast differentiation in fin regenerates. Together, these data indicate that bone healing in zebrafish fin regeneration was improved by activating Wnt signaling using GSK3b inhibitor treatment. In addition, caudal fin regeneration is useful to evaluate dose-dependent pharmacological efficacy in bone healing, various dosing regimens and possible toxicological effects of compounds.
Wnt信号通路已被证明可调节骨稳态并影响某些骨疾病状态。我们利用斑马鱼模型系统研究了一种合成的、口服生物可利用的糖原合酶激酶-3β（GSK3β）抑制剂LSN 2105786的作用，该抑制剂在骨愈合和胚胎发育过程中激活Wnt信号。使用GSK3β抑制剂处理来模拟斑马鱼胚胎中GSK3β吗啉代寡核苷酸（MO）敲低的效果。人源和斑马鱼源合成mRNA注射在挽救GSK3β MO敲低方面同样有效。在尾鳍再生过程中，骨射线是斑马鱼鳍中第一个分化的结构，为研究骨愈合提供了一个有用的模型。使用不同浓度的GSK3β抑制剂进行尾鳍再生实验，研究再生生长的持续时间和浓度依赖性。实验表明，持续低浓度（4-5 nM）处理在促进再生方面比间歇给药更有效。较高浓度会抑制鳍的生长，可能是由于过度刺激分化程序。观察到GSK3β抑制剂处理后Wnt反应性基因表达和分化增加。激活Wnt信号还增加了鳍再生中的细胞增殖和成骨细胞分化。总之，这些数据表明，使用GSK3β抑制剂处理激活Wnt信号可改善斑马鱼鳍再生中的骨愈合。此外，尾鳍再生对于评估骨愈合中化合物的剂量依赖性药理疗效、各种给药方案和可能的毒理学效应很有用。
