Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

尽管关节软骨（articular cartilage）退变是一类重大公共卫生问题，但其潜在分子机制仍未得到充分阐明。我们此前曾利用全基因组表达分析（genome-wide expression analysis）鉴定出猪关节软骨的特异性标志物，其中之一为血小板反应蛋白4（Thrombospondin-4, Thbs4）。本研究中，我们对Thbs4在小鼠体内的表达模式进行了分析，不仅证实其在关节软骨中的优势表达，还发现其在包括骨骼在内的其他组织中亦有表达。
为探究Thbs4在骨骼发育与稳态维持中的作用，我们借助Thbs4基因敲除小鼠模型，通过非脱钙骨组织学（undecalcified bone histology）分析开展研究。结果显示，与野生型同窝小鼠（wildtype littermates）相比，Thbs4敲除小鼠的骨骼生长及骨量获取均未表现出显著表型差异。鉴于此前研究发现Thbs4在Phex基因敲除的Hyp小鼠（Phex-deficient Hyp mice）骨骼中呈过表达状态，我们额外构建了Thbs4敲除的Hyp小鼠，但同样未观察到其与Hyp型同窝小鼠存在表型差异。
针对关节软骨的分析显示，Thbs4敲除小鼠表现出关节软骨的一过性变薄，提示Thbs4对关节稳态具有保护作用。利用猪原代细胞进行的基因表达分析表明，滑膜成纤维细胞（synovial fibroblasts）不表达Thbs4，且Thbs4是血小板反应蛋白基因家族中唯一在关节软骨而非生长板软骨细胞（growth plate chondrocytes）中特异性表达的成员。
为进一步明确Thbs4的特异性分子效应，我们在有无猪滑膜成纤维细胞条件培养基（conditioned medium）的情况下，用重组人THBS4处理猪关节软骨细胞（porcine articular chondrocytes）。结果未观察到THBS4对细胞增殖（proliferation）、代谢活性（metabolic activity）、细胞凋亡（apoptosis）或基因表达产生显著影响，提示其并非作为信号分子发挥作用。
综上，本研究数据证实Thbs4在关节软骨细胞中高表达，且其在细胞外基质（extracellular matrix）中的存在对维持关节软骨稳态至关重要。