Nishioka Katsuhiro, Itoh Shunji, Suemoto Hiroki, Kanno Seiji, Gai Zhibo, Kawakatsu Motohisa, Tanishima Hiroyuki, Morimoto Yoshifumi, Hatamura Ikuji, Yoshida Munehito, Muragaki Yasuteru
First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
Department of Orthopedic Surgery, Wakayama Medical University School of Medicine, Wakayama, 641-0012, Japan.
Bone. 2008 Jul;43(1):64-71. doi: 10.1016/j.bone.2008.03.009. Epub 2008 Mar 29.
We have reported that elongation of the columnar proliferative zone of long bone growth plates in Trps1-/- mice during the late fetal stage in the previous study [1]. Since expression of Trps1 protein was found to overlap with that of mRNAs for Indian hedgehog (Ihh), PTH/PTHrP receptor (PPR), and PTHrP, we hypothesized that Trps1 may inhibit the hypertrophic differentiation of chondrocytes by interacting with the Ihh/PTHrP feedback loop. To investigate whether Trps1 has a role in this Ihh/PTHrP feedback loop, we compared the growth plates of Trps1-/- mice and wild-type (Trps1+/+) mice. Immunohistochemistry showed that Trps1 protein was strongly expressed in the periarticular and prehypertrophic zones of the fetal growth plate in wild-type mice on embryonic day 18.5 (E18.5). On the other hand, Ihh, PPR, and PTHrP mRNAs were predominantly expressed in the prehypertrophic zone at this stage of development. While expression of Ihh and PPR by prehypertrophic chondrocytes was unaffected in the growth plates of Trps1-/- mice, the range of PTHrP expression was expanded toward the proliferating zone in these mice. Quantitative real-time PCR analysis demonstrated upregulation of PTHrP in the epiphyseal growth plates of Trps1-/- mice. Furthermore, promoter analysis combined with the chromatin immunoprecipitation (ChIP) assay demonstrated that direct binding of Trps1 to the PTHrP promoter suppressed the transcription of PTHrP. Finally, organ culture of E14.5 tibiae in the absence or the presence of Pthrp revealed that the proliferative zone of the tibial growth plate was elongated by culture with Pthrp compared to that of control tibiae. Taken together, these data provide the first genetic evidence that lack of Trps1 leads to overexpression of PTHrP, and that Trps1 is required to maintain the normal organization of chondrocytes in the growth plate.
我们在之前的研究中报道过，在胎儿晚期，Trps1基因敲除（Trps1-/-）小鼠长骨生长板柱状增殖区会延长[1]。由于发现Trps1蛋白的表达与印度刺猬因子（Ihh）、甲状旁腺激素/甲状旁腺激素相关蛋白受体（PPR）和甲状旁腺激素相关蛋白（PTHrP）的mRNA表达重叠，我们推测Trps1可能通过与Ihh/PTHrP反馈环相互作用来抑制软骨细胞的肥大分化。为了研究Trps1在这个Ihh/PTHrP反馈环中是否起作用，我们比较了Trps1-/-小鼠和野生型（Trps1+/+）小鼠的生长板。免疫组织化学显示，在胚胎第18.5天（E18.5），野生型小鼠胎儿生长板的关节周围和肥大前区强烈表达Trps1蛋白。另一方面，在这个发育阶段，Ihh、PPR和PTHrP的mRNA主要在肥大前区表达。虽然Trps1-/-小鼠生长板中肥大前软骨细胞的Ihh和PPR表达未受影响，但这些小鼠中PTHrP的表达范围向增殖区扩展。定量实时PCR分析表明，Trps1-/-小鼠骨骺生长板中PTHrP上调。此外，启动子分析结合染色质免疫沉淀（ChIP）试验表明，Trps1与PTHrP启动子的直接结合抑制了PTHrP的转录。最后，在有无Pthrp的情况下对E14.5胫骨进行器官培养，结果显示与对照胫骨相比，用Pthrp培养可使胫骨生长板的增殖区延长。综上所述，这些数据提供了首个遗传学证据，表明Trps1的缺失导致PTHrP过表达，且Trps1是维持生长板中软骨细胞正常组织所必需的。
