Role of the transcriptional regulator AP-2 in chondrocytes

最近，我们发现了转录因子AP-2 epsilon在肥大和关节软骨组织中的蛋白表达。此外，还检测了骨关节炎(OA)患者软骨细胞中AP-2 epsilon mRNA的表达增强。在本项目的第一个资助期，可以确定软骨中AP-2 epsilon的两个靶基因：CXCL1和II型胶原基因。这些数据被成功地发表在两种科学期刊上。此外，我们分析了代表肢体发育不同阶段的WT小鼠的软骨组织样本中AP-2 epsilon的表达。观察到转录因子的表达水平被强烈诱导。许多实验证明，低氧而不是机械负荷是这一调节过程的关键外部刺激。目前，这些数据正在提交以供发布。到目前为止，对AP-2 epsilon缺陷胎儿在不同时间点的胚胎软骨和骨骼发育的分析显示，与WT动物没有或仅有微小的差异。这可能是由于AP-2 epsilon在早期胚胎软骨结构和/或代偿机制中相对较低的表达。为了确定AP-2 epsilon在高度分化的关节软骨中的作用以及在骨关节炎的发生发展过程中的作用，我们对成年小鼠进行了骨关节炎的体内模型研究。在这里，与WT相比，AP-2 epsilon缺陷动物在OA诱导17天后关节软骨表面的损伤量显著增加。有趣的是，在缺乏AP-2 epsilon的软骨中，基质金属蛋白酶Mmp13的活性显著增强，这为在基因敲除小鼠中观察到的异常疾病进展提供了合理的解释。在本项目的更新中，将进一步分析AP-2 epsilon对成人关节软骨内稳态的影响。特别令人感兴趣的是解释在AP-2 epsilon缺陷小鼠中观察到的Mmp13异常的分子机制。另一个目标是通过微阵列实验发现更多在AP-2 epsilon基因敲除标本的关节软骨中异常表达的基因。此外，AP-2 epsilon在早期胚胎发育过程中过度表达的转基因小鼠模型应该为转录因子在骨骼发育中的作用提供有价值的信息。此外，还计划使用AP-2 epsilon启动子驱动的GFP小鼠模型，详细分析AP-2 epsilon在胚胎发育到完全分化的关节软骨过程中的生理性表达。同时，该模型和相应的体外实验将有助于确定在低氧之后调节AP-2 epsilon表达的其他潜在因素。这些实验结果将有助于对关节软骨的软骨形成和致病过程有更全面的了解。

Recently, we revealed protein expression of the transcription factor AP-2 epsilon in hypertrophic and articular cartilage tissue. Additionally, enhanced AP-2 epsilon mRNA expression in chondrocytes of osteoarthritis (OA) patients was determined. In the first funding period of this project two target genes of AP-2 epsilon in cartilage could be identified: CXCL1 as well as the gene for type II collagen. These data were published successfully in two scientific journals. In addition, we analyzed cartilaginous tissue samples of WT mice representing different stages of limb development for AP-2 epsilon expression. A strong induction of the expression level of the transcription factor was observed. A number of experiments provided evidence that hypoxia but not mechanical loading is a crucial external stimulus for this regulatory process. Currently, these data are submitted for publication. So far, analysis of embryonic cartilage and skeletal development in AP-2 epsilon deficient feti at various time points revealed no or just minor discrepancies to WT animals. This might be due to relatively low AP-2 epsilon expression in early embryonic cartilage structures and/or compensatory mechanisms.To determine the role of AP-2 epsilon in highly differentiated, articular cartilage as well as during osteoarthritis development and progression adult mice were subjected to an in vivo model for osteoarthritis. Here, the amount of damage to the articular cartilage surface was significantly increased in the AP-2 epsilon deficient animals 17 days after OA induction compared to the WT. Interestingly, activity of the matrix metalloproteinase Mmp13 was significantly enhanced in cartilage lacking AP-2 epsilon, offering a reasonable explanation for the abnormal disease progression observed in the knock-out mice. Within the renewal of this project the influence of AP-2 epsilon on the homeostasis of adult articular cartilage will be analyzed further. Of special interest is the molecular mechanism accounting for the observed Mmp13 dysregulation in AP-2 epsilon deficient mice. Another goal is to identify more genes that exhibit abnormal expression in articular cartilage of AP-2 epsilon knock-out specimen via microarray experiments. Additionally, a transgenic mouse model for AP-2 epsilon over-expression during early embryogenesis should provide valuable information about the role of the transcription factor during skeletal development. Furthermore, an in detail analysis of physiological AP-2 epsilon expression during embryonic development up to fully differentiated articular cartilage using an AP-2 epsilon promoter driven GFP mouse model is planned. Simultaneously, this model and corresponding in vitro experiments will be useful to identify additional potential factors regulating AP-2 epsilon expression next to hypoxia. The results of these experiments will contribute to the general understanding of chondrogenesis and pathogenic processes in articular cartilage.