Role of the transcriptional regulator AP-2 in chondrocytes

最近，我们发现在肥大和关节软骨组织中有转录因子AP-2 β的蛋白表达。此外，还测定了骨关节炎（OA）患者软骨细胞中AP-2 mRNA表达的增强。在该项目的第一个资助期内，可以确定软骨中AP-2的两个靶基因：CXCL 1以及II型胶原基因。这些数据成功地发表在两份科学期刊上。 此外，我们分析了代表不同肢体发育阶段的WT小鼠软骨组织样品的AP-2 β表达。观察到转录因子表达水平的强烈诱导。许多实验提供的证据表明，缺氧，而不是机械负荷是一个至关重要的外部刺激，这一监管过程。目前，这些数据已提交出版。到目前为止，在不同时间点对AP-2 β缺陷胎儿的胚胎软骨和骨骼发育的分析显示与WT动物没有差异或只有微小差异。这可能是由于早期胚胎软骨结构和/或代偿机制中AP-2 β表达相对较低。为了确定AP-2 β在高度分化的关节软骨以及骨关节炎发展和进展过程中的作用，对成年小鼠进行骨关节炎的体内模型。在此，与WT相比，在OA诱导后17天，AP-2 β缺陷动物中关节软骨表面的损伤量显著增加。有趣的是，基质金属蛋白酶Mmp 13的活性在缺乏AP-2 β的软骨中显著增强，为在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对成人关节软骨稳态的影响。特别令人感兴趣的是在AP-2缺陷小鼠中观察到的Mmp 13失调的分子机制。另一个目标是通过基因芯片实验来鉴定更多在AP-2基因敲除标本的关节软骨中表现出异常表达的基因。此外，在早期胚胎发育过程中AP-2过表达的转基因小鼠模型应该提供有关转录因子在骨骼发育过程中的作用的有价值的信息。此外，计划使用AP-2 β启动子驱动的GFP小鼠模型详细分析胚胎发育直至完全分化的关节软骨期间的生理AP-2 β表达。同时，该模型和相应的体外实验将有助于确定其他潜在的因素调节AP-2 β表达缺氧。这些实验的结果将有助于对关节软骨中的软骨发生和致病过程的一般理解。

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.