C-type natriuretic peptide regulates endochondral ossification through p38 MAP kinase-dependent pathways_2. Mus musculus

C-type natriuretic peptide (CNP) has been recently identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanism mediating these effects are not completely understood. Here we demonstrate that CNP activates the p38 MAP kinase pathway in chondrocytes and that pharmacological inhibition of p38 blocks the anabolic effects of CNP in a tibia organ culture system. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We performed Affymetrix microarray analyses to identify CNP target genes in the organ culture system. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, potentially because cGMP-dependent kinases I and II, important transducers of CNP signaling and are expressed at much higher levels in these cells than in other areas of the tibia. One of the genes most strongly induced by CNP was the Ptgs2 gene, encoding Cox2. Real-time PCR confirmed that Cox2 expression was induced by CNP in hypertrophic chondrocytes, but surprisingly in a p38-independent manner. Moreover, Cox2 inhibition – in contrast to p38 inhibition - did not block the anabolic effects of CNP. In summary, our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral ossification, with potential implications for numerous skeletal diseases. Keywords: Growth plate zone comparison and treatment response analysis Overall design: Tibiae from E15.5 day old embryonic mice were isolated and cultured in minimal media in the presence of vehicle, BSA/HCl (1mM), or C-type natriuretic peptide, CNP (10-6M). On the sixth day of treatment cultured tibias were micro-dissected into the resting/proliferating, hypertrophic, and mineralized areas. Distinct zones from approximately 24 bones were pooled together, from which RNA was isolated using the Qiagen RNeasy Lipid Extraction Kit. Once the quality of total RNA from three independent trials was determined using the Agilent 2100 bioanalyzer, microarray analyses were performed at the London Regional Genomics Centre using MOE430_2.0 Affymetrix arrays. Results were analyzed using GeneSpring 7.2 software.

C型利钠肽（C-type natriuretic peptide, CNP）近来被鉴定为软骨内骨生长的重要合成代谢调控因子，但其介导此类效应的分子机制尚未完全阐明。本研究证实，C型利钠肽可激活软骨细胞内的p38丝裂原活化蛋白激酶（p38 MAP kinase）通路，而p38的药理学抑制可在胫骨器官培养体系中阻断C型利钠肽的合成代谢效应。我们进一步发现，C型利钠肽主要通过扩大生长板的肥大区刺激软骨内骨生长，同时延缓矿化过程；上述两种效应均可被p38抑制所逆转。我们通过Affymetrix基因芯片（Affymetrix）分析，鉴定了该器官培养体系中CNP的靶基因。上述研究证实，肥大软骨细胞是生长板中CNP信号的主要靶标，这可能是因为作为CNP信号重要转导分子的cGMP依赖性蛋白激酶I和II（cGMP-dependent kinases I and II），在该类细胞中的表达水平远高于胫骨其他区域。受CNP诱导最显著的基因之一为Ptgs2基因，其编码环氧合酶2（Cox2）。实时荧光定量PCR（Real-time PCR）证实，CNP可在肥大软骨细胞中诱导Cox2的表达，但令人意外的是，该诱导过程不依赖于p38通路。此外，与p38抑制不同，Cox2抑制并未阻断CNP的合成代谢效应。综上，本研究鉴定了CNP的新型靶基因，并证实p38通路是介导CNP调控软骨内骨化的新型必需介质，该发现对多种骨骼疾病具有潜在研究价值。
关键词：生长板区域比较与治疗应答分析
整体实验设计：将15.5天胚胎期（E15.5）小鼠的胫骨分离后，置于基础培养基中培养，分别添加溶剂对照、牛血清白蛋白（BSA）/盐酸（HCl，1mM）或C型利钠肽（CNP，10^-6 M）。给药处理第6天，将培养的胫骨显微解剖为静息/增殖区、肥大区及矿化区。将约24根骨骼的不同区域组织混合收集，使用凯杰（Qiagen）RNeasy脂质提取试剂盒提取总RNA。通过安捷伦2100生物分析仪（Agilent 2100 bioanalyzer）对三次独立重复实验的总RNA质量进行检测后，使用MOE430_2.0型Affymetrix基因芯片，在伦敦区域基因组学中心完成基因芯片分析。实验结果采用GeneSpring 7.2软件进行分析。