The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

肌腱中的生物钟调节 I 型胶原蛋白的日常节律合成以及细胞外基质中小直径胶原纤维的出现和消失。原纤维如何组装和去除尚不完全清楚。在这里，我们首先表明胶原酶，即膜型 I 基质金属蛋白酶（MT1-MMP，由Mmp14编码），受到出生后小鼠肌腱中生物钟的调节。接下来，我们生成了他莫昔芬诱导的Col1a2-Cre-ERT2::Mmp14 KO 小鼠（ Mmp14条件敲除 (CKO)）。 CKO小鼠出现后肢背屈和肌腱增厚，这些肌腱积累了窄直径的胶原纤维，导致超微结构紊乱。对照肌腱的质谱分析鉴定出 1195 种蛋白质，其中 212 种显示出时间依赖性丰度。在Mmp14 CKO 小鼠中，19 种蛋白质的时间丰度发生逆转，176 种蛋白质失去了时间依赖性。其中，胶原交联酶赖氨酰氧化酶样 1 (LOXL1) 和赖氨酰羟化酶 1 (LH1；由Plod2编码) 升高并失去时间依赖性调节。高压色谱法证实 CKO 肌腱中胶原蛋白的羟赖氨酸醛（吡啶啉）交联水平升高。结果，I 型胶原蛋白难以提取。我们还表明，CRISPR-Cas9从培养的成纤维细胞中删除Mmp14会导致第2周期（PER2）生物钟节律的丧失，而重组MT1-MMP在裂解可溶性胶原蛋白-I方面非常有效，但在裂解预组装的胶原蛋白方面效果较差成原纤维。 总之，我们的研究表明，生物钟调节的Mmp14控制小直径胶原纤维的节律合成，调节胶原交联，并且它的缺失会扰乱肌腱成纤维细胞中的生物钟和基质体。