García-Martín A, Ardura J A, Maycas M, Lozano D, López-Herradón A, Portal-Núñez S, García-Ocaña A, Esbrit P
Laboratorio de Metabolismo Mineral y Óseo (A.G-M., J.A.A., M.M., D.L., A.L-H., S.P-N., P.E.), Instituto de Investigación Sanitaria (IIS)-Fundación Jiménez Díaz and Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Instituto de Salud Carlos III, Madrid, 28040, Spain; and Department of Medicine (A.G-O.), Division of Endocrinology and Metabolism, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
Mol Endocrinol. 2014 Jun;28(6):925-34. doi: 10.1210/me.2013-1225. Epub 2014 Apr 11.
PTHrP is an important regulator of bone remodelling, apparently by acting through several sequence domains. We here aimed to further delineate the functional roles of the nuclear localization signal (NLS) comprising the 88-107 amino acid sequence of PTHrP in osteoblasts. PTHrP mutants from a human PTHrP (-36/+139) cDNA (wild type) cloned into pcDNA3.1 plasmid with deletion (Δ) of the signal peptide (SP), NLS, T(107), or T107A replacing T(107) by A(107) were generated and stably transfected into osteoblastic MC3T3-E1 cells. In these cells, intracellular trafficking, cell proliferation and viability, as well as cell differentiation were evaluated. In these transfected cells, PTHrP was detected in the cytoplasm and also in the nucleus, except in the NLS mutant. Meanwhile, the PTH type 1 receptor (PTH1R) accumulates in the cytoplasm except for the ΔSP mutant in which the receptor remains at the cell membrane. PTHrP-wild type cells showed enhanced growth and viability, as well as an increased matrix mineralization, alkaline phosphatase activity, and osteocalcin gene expression; and these features were inhibited or abolished in ΔNLS or ΔT(107) mutants. Of note, these effects of PTHrP overexpression on cell growth and function were similarly decreased in the ΔSP mutant after PTH1R small interfering RNA transfection or by a PTH1R antagonist. The present in vitro findings suggest a mixed model for PTHrP actions on osteoblastic growth and function whereby this protein needs to be secreted and internalized via the PTH1R (autocrine/paracrine pathway) before NLS-dependent shuttling to the nucleus (intracrine pathway).
甲状旁腺激素相关蛋白（PTHrP）显然是通过几个序列结构域发挥作用，是骨重塑的重要调节因子。我们的目的是进一步阐明甲状旁腺激素相关蛋白（PTHrP）中包含88 - 107个氨基酸序列的核定位信号（NLS）在成骨细胞中的功能作用。从克隆到pcDNA3.1质粒中的人PTHrP（-36 / +139）cDNA（野生型）产生了信号肽（SP）、NLS、T（107）缺失或T107A（用A（107）取代T（107））的PTHrP突变体，并将其稳定转染到成骨MC3T3 - E1细胞中。在这些细胞中，评估了细胞内运输、细胞增殖和活力以及细胞分化。在这些转染细胞中，除了NLS突变体，在细胞质和细胞核中均检测到PTHrP。同时，1型甲状旁腺激素受体（PTH1R）在细胞质中积累，除了ΔSP突变体，该突变体中的受体保留在细胞膜上。PTHrP野生型细胞显示出增强的生长和活力，以及增加的基质矿化、碱性磷酸酶活性和骨钙素基因表达；而这些特征在ΔNLS或ΔT（107）突变体中受到抑制或消除。值得注意的是，在PTH1R小干扰RNA转染后或使用PTH1R拮抗剂处理后，ΔSP突变体中PTHrP过表达对细胞生长和功能的这些影响同样降低。目前的体外研究结果提示了一种PTHrP对成骨细胞生长和功能作用的混合模型，即该蛋白需要先通过PTH1R分泌并内化（自分泌/旁分泌途径），然后通过依赖NLS的方式穿梭进入细胞核（内分泌途径）。
