Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
J Mol Endocrinol. 2013 Jun 18;51(1):99-108. doi: 10.1530/JME-12-0251. Print 2013.
Osterix (Osx, Sp7) is a zinc-finger transcription factor belonging to the specificity protein (Sp) family expressed in cells of the osteoblast lineage in the developing skeleton where it regulates expression of a number of osteoblastic genes. We previously reported inhibition of osterix mRNA and protein by parathyroid hormone (PTH) stimulation of cAMP in osteoblasts. We here show that Osx expression in osteoblasts is regulated by Sp proteins as demonstrated by mithramycin A inhibition of Osx mRNA and OSX protein levels. Mutation of putative transcription factor binding sites within the Osx promoter demonstrated a tandem repeat sequence that selectively binds OSX but not other Sp factors expressed in osteoblasts (Sp1, Sp3, or Tieg (Klf10)). Mutation of either or both the repeat sequences inhibited 90% of the promoter activity and also abrogated some of the PTH-mediated inhibition of the promoter. Previous studies have shown growth factor regulation of Osx expression by MAPK proteins, particularly p38 phosphorylation of OSX that increases its transcriptional activity. PTH stimulation of osteoblasts inhibits MAPK components (ERK, JNK, and p38) but inhibition of Osx mRNA and protein expression by PTH was selectively mimicked by p38 inhibition and expression of constitutively active MKK6, which stimulates p38, blocked PTH inhibition of OSX. Together, our studies suggest that OSX autoregulation is a major mechanism in osteoblasts and that PTH stimulation inhibits osterix by inhibition of p38 MAPK regulation of OSX.
成骨细胞特异性转录因子 2（osterix，Osx，Sp7）属于碱性螺旋-环-螺旋转录因子超家族，特异性表达于正在发育的骨骼中处于成骨细胞谱系的细胞，调节许多成骨细胞基因的表达。我们之前的研究表明，甲状旁腺激素（PTH）通过刺激细胞内环磷酸腺苷（cAMP）抑制成骨细胞中 osterix mRNA 和蛋白的表达。我们在此证明，成骨细胞中 Osx 的表达受 Sp 蛋白调节，米托蒽醌 A 抑制 Osx mRNA 和 OSX 蛋白水平证实了这一点。Osx 启动子中假定转录因子结合位点的突变显示串联重复序列，该序列选择性结合 OSX，但不结合成骨细胞中表达的其他 Sp 因子（Sp1、Sp3 或 Tieg（Klf10））。重复序列的突变或两者的突变均抑制了 90%的启动子活性，并部分消除了 PTH 对启动子的抑制作用。先前的研究表明生长因子通过丝裂原活化蛋白激酶（MAPK）蛋白调节 Osx 的表达，特别是 OSX 的 p38 磷酸化增加其转录活性。PTH 刺激成骨细胞抑制 MAPK 成分（ERK、JNK 和 p38），但 p38 抑制选择性模拟了 PTH 对 Osx mRNA 和蛋白表达的抑制作用，而表达组成型激活的 MKK6 刺激 p38，可阻断 PTH 对 OSX 的抑制作用。总之，我们的研究表明 OSX 自身调节是成骨细胞中的主要机制，而 PTH 刺激通过抑制 p38 MAPK 对 OSX 的调节来抑制 osterix。
