Olivares-Navarrete R, Vesper K, Hyzy S L, Almaguer-Flores A, Boyan B D, Schwartz Z
Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, 601 West Main Street, Suite 396, Richmond, VA 23284,
Eur Cell Mater. 2014 Jul 14;28:1-10; discussion 10. doi: 10.22203/ecm.v028a01.
Porcine enamel matrix derivative (pEMD), a complex mixture of proteins and peptides including full-length amelogenin protein, splice variants, and proteolytic peptides, is used clinically with a carrier to regenerate supportive tissue around teeth. During application, pEMD self-assembles as nanospheres and precipitates as a three-dimensional matrix to facilitate cell migration and differentiation. Amelogenin, the primary constituent of pEMD, stimulates osteoblast differentiation, but it is unclear what specific roles other components of pEMD play in determining biological response. This study examined the potential of one constituent of pEMD, the N-terminal amelogenin peptide (NTAP), to promote osteoblastic differentiation of human mesenchymal stem cells (MSCs) and to elucidate possible signaling pathways involved. Effects of porcine NTAP on MSC cultures were compared to those of recombinant human amelogenin. While amelogenin induced MSC osteoblastic differentiation, a more robust osteoblastic response was seen after NTAP treatment. A phospho-kinase proteasome array measuring phosphorylation of 35 proteins indicated that protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and β-catenin were highly phosphorylated by NTAP. This was confirmed by measuring PKC activity and levels of phospho-ERK1/2 and β-catenin. Both amelogenin and NTAP increased PKC, but NTAP induced higher phosho-ERK1/2 and phospho-β-catenin than amelogenin. ERK1/2 inhibition blocked both amelogenin- and NTAP-induced increases in RUNX2, ALP, OCN, COL1, and BMP2. The results demonstrate that NTAP induces osteogenic differentiation of MSCs via PKC and ERK1/2 activation and β-catenin degradation. NTAP may be an active bone regeneration component of amelogenin, and may play this role in pEMD-stimulated periodontal regeneration.
猪牙釉质基质衍生物（pEMD）是一种蛋白质和肽的复杂混合物，包括全长釉原蛋白、剪接变体和蛋白水解肽，临床上与载体一起用于再生牙齿周围的支持组织。在应用过程中，pEMD自组装成纳米球并沉淀为三维基质，以促进细胞迁移和分化。釉原蛋白是pEMD的主要成分，可刺激成骨细胞分化，但尚不清楚pEMD的其他成分在决定生物学反应中起什么具体作用。本研究检测了pEMD的一种成分N端釉原蛋白肽（NTAP）促进人间充质干细胞（MSC）成骨分化的潜力，并阐明了可能涉及的信号通路。将猪NTAP对MSC培养物的影响与重组人釉原蛋白的影响进行了比较。虽然釉原蛋白诱导了MSC的成骨分化，但NTAP处理后观察到了更强的成骨反应。一项测量35种蛋白质磷酸化的磷酸激酶蛋白酶体阵列表明，蛋白激酶C（PKC）、细胞外信号调节激酶1/2（ERK1/2）和β-连环蛋白被NTAP高度磷酸化。通过测量PKC活性以及磷酸化ERK1/2和β-连环蛋白的水平证实了这一点。釉原蛋白和NTAP均增加了PKC，但NTAP诱导的磷酸化ERK1/2和磷酸化β-连环蛋白高于釉原蛋白。ERK1/2抑制阻断了釉原蛋白和NTAP诱导RUNX2、ALP、OCN、COL1和BMP2的增加。结果表明，NTAP通过PKC和ERK1/2激活以及β-连环蛋白降解诱导MSC的成骨分化。NTAP可能是釉原蛋白的一种活性骨再生成分，并且可能在pEMD刺激的牙周再生中发挥这一作用。
