Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States of America.
Department of Health Sciences Research, Mayo Clinic, Rochester, MN, United States of America.
Bone. 2024 Dec;189:117265. doi: 10.1016/j.bone.2024.117265. Epub 2024 Sep 29.
Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2 mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2 (double knockout) mice resembled Phlpp1 mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1 and Phlpp1/2 chondrocytes had similarities and were significantly different from WT and Phlpp2 chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.
附肢骨骼的生长和骨量的获得是由多种生长因子、激素和机械力在一个称为软骨内骨化的动态过程中控制的。在长骨中，生长板中的软骨细胞增殖并发生肥大，从而驱动骨的伸长和矿化。Pleckstrin 同源（PH）结构域和富含亮氨酸重复的磷酸酶 1 和 2（Phlpp1 和 Phlpp2）是丝氨酸/苏氨酸蛋白磷酸酶，通过 Akt、PKC、Raf1、S6k 和其他细胞内信号级联调节细胞增殖、存活和成熟。Phlpp1 的种系缺失抑制生长板软骨细胞的骨伸长。在这里，我们证明 Phlpp2 不调节软骨内骨化，并且我们定义了软骨细胞中 Phlpp1 和 Phlpp2 之间的分子差异。Phlpp2 小鼠与其野生型（WT）同窝仔鼠表型上无法区分，具有相似的骨长度、骨量和生长板动力学。与 WT 细胞相比，Phlpp2 的缺失对软骨细胞转录组和蛋白质组有适度的影响。相比之下，Phlpp1/2（双敲除）小鼠在表型和分子上与 Phlpp1 小鼠相似，因为 Phlpp1 和 Phlpp1/2 软骨细胞的磷酸化蛋白组具有相似性，并且与 WT 和 Phlpp2 软骨细胞的磷酸化蛋白组显著不同。通过多参数分析进行的数据整合表明，与蛋白质组或磷酸化蛋白质组相比，由于 Phlpp1 或 Phlpp2 缺失导致的转录组解释数据变化较小。在缺乏 Phlpp1 的软骨细胞中鉴定到细胞增殖、胶原纤维组织和 Pdpk1 和 Pak1/2 信号通路的改变，而在缺乏 Phlpp2 的软骨细胞中改变了细胞周期过程和 Akt1 和 Aurka 信号通路。这些数据表明，Phlpp1（在较小程度上 Phlpp2）调节跨软骨细胞转录组、蛋白质组和磷酸化蛋白质组的多个复杂信号级联，并且多组学数据整合可以揭示调节软骨内骨化的新潜在激酶靶标。
