Killinger Michael, Szotkowská Tereza, Lusková Denisa, Zezula Nikodém, Bryja Vítězslav, Buchtová Marcela
Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 602 00 Brno, Czech Republic.
Department of Experimental Biology, Faculty of Sciences, Masaryk University, 62504 Brno, Czech Republic.
JBMR Plus. 2025 Mar 29;9(6):ziaf048. doi: 10.1093/jbmrpl/ziaf048. eCollection 2025 Jun.
Porcupine (PORCN) is a membrane-bound protein of the endoplasmic reticulum, which modifies Wnt proteins by adding palmitoleic acid. This modification is essential for Wnt ligand secretion. Patients with mutated PORCN display various skeletal abnormalities likely stemming from disrupted Wnt signaling pathways during the chondrocyte differentiation. To uncover the mechanism of PORCN action during chondrogenesis, we used 2 different PORCN inhibitors, C59 and LGK974, in several model systems, including micromasses, 3D cell cultures, long bone tissue cultures, and zebrafish animal model. PORCN inhibitors enhanced cartilaginous extracellular matrix (ECM) production and accelerated chondrocyte differentiation, which resulted in the earlier induction of cellular hypertrophy as well as cartilaginous mass expansion in micromass cultures and cartilaginous organoids. In addition, both PORCN inhibitors expanded the hypertrophic zone and reduced the proliferative zone in the growth plate. This led to a significant increase in cartilaginous tissue and ultimately resulted in the elongation of tibias in the mouse organ cultures. Also, LGK974 treatment of embryos induced expansion of craniofacial cartilage width together with the shortening of the body axis, which was consistent with a phenomenon occurring upon inhibition of non-canonical Wnt signaling. By combining PORCN inhibition with exogenous Wnt proteins activating either canonical/β-catenin (WNT3a) or non-canonical (WNT5a) signaling, we propose that the key mechanism mediating pro-chondrogenic effects of PORCN inhibition is the removal of canonical ligands that prevent chondrocyte differentiation. In summary, our results provide evidence of the distinct role of PORCN in both the early and late stages of cartilage development. Further, our data demonstrate that PORCN inhibitors can be used in the experimental and clinical strategies that need to trigger chondrocyte differentiation and/or cartilage outgrowth.
刺猬蛋白（PORCN）是一种内质网的膜结合蛋白，它通过添加棕榈油酸来修饰Wnt蛋白。这种修饰对于Wnt配体的分泌至关重要。PORCN发生突变的患者表现出各种骨骼异常，这可能源于软骨细胞分化过程中Wnt信号通路的中断。为了揭示PORCN在软骨形成过程中的作用机制，我们在几种模型系统中使用了两种不同的PORCN抑制剂C59和LGK974，包括微团培养、三维细胞培养、长骨组织培养和斑马鱼动物模型。PORCN抑制剂增强了软骨细胞外基质（ECM）的产生并加速了软骨细胞的分化，这导致在微团培养和软骨类器官中更早地诱导细胞肥大以及软骨块扩张。此外，两种PORCN抑制剂都扩大了生长板中的肥大区并减少了增殖区。这导致软骨组织显著增加，并最终导致小鼠器官培养物中胫骨的伸长。同样，用LGK974处理胚胎会诱导颅面软骨宽度增加以及身体轴缩短，这与抑制非经典Wnt信号时出现的现象一致。通过将PORCN抑制与激活经典/β-连环蛋白（WNT3a）或非经典（WNT5a）信号的外源性Wnt蛋白相结合，我们提出介导PORCN抑制的促软骨生成作用的关键机制是去除阻止软骨细胞分化的经典配体。总之，我们的结果提供了PORCN在软骨发育的早期和晚期具有不同作用的证据。此外，我们的数据表明PORCN抑制剂可用于需要触发软骨细胞分化和/或软骨生长的实验和临床策略。
