Morrison D F, Mauro L J
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Gene. 2000 Oct 31;257(2):195-208. doi: 10.1016/s0378-1119(00)00397-8.
Tyrosine kinases and phosphatases are regulators of the steady-state levels of phosphotyrosine proteins and, in this way, are key players in determining the functional state of the cell. As a unique member of the protein tyrosine phosphatase (PTP) superfamily, osteotesticular PTP (OST-PTP) is a receptor protein whose expression is highly regulated during osteoblast differentiation and in response to modulators of bone remodeling such as parathyroid hormone and vitamin D3. To explore the molecular mechanisms and signaling pathways important in the regulation of this gene, we characterized the structural organization of the mouse OST-PTP cDNA and gene and determined its chromosomal localization. The mouse cDNA is approximately 5.5 kb including 5.1 kb of coding sequence, 315 bp 5' UTR and 102 bp 3' UTR. It is expressed as a single approximately 5.8 kb transcript in day 8 differentiated MC3T3 osteoblasts. Although highly homologous to the rat OST-PTP cDNA, the mouse cDNA possesses a 74 bp insert in the 5' UTR which contains several potential transcription factor binding sites such as AP-2 and NFkappaB. The mouse OST-PTP (mOST-PTP) gene is a single copy gene encompassing 35 exons and spanning only 20.65 kb. As such, it is the smallest gene of the characterized receptor PTP genes. This is due to the lack of large introns and the conserved spatial organization of exons which encode specific protein motifs in the mOST-PTP molecule. Sequence analysis of the putative mOST-PTP promoter revealed basal elements as well as many potential cis-acting regulatory elements with relevance to gene regulation in bone. Of particular interest is the single osteoblast specific element known as osteocalcin specific element 2 (OSE2) found at position -1867, as well as numerous VDRE and NFkappaB sites found throughout the promoter and the 5' UTR. Fluorescence in situ hybridization studies have shown that mOST-PTP localizes to mouse chromosome 1, region F-G which is syntenic to the segment of human chromosome 1q32-33. This characterization of the mOST-PTP cDNA and gene will facilitate future experiments exploring the mechanisms of regulation of this phosphatase during osteogenesis.
酪氨酸激酶和磷酸酶是磷酸化酪氨酸蛋白稳态水平的调节因子，因此是决定细胞功能状态的关键因素。作为蛋白酪氨酸磷酸酶（PTP）超家族的独特成员，骨睾丸磷酸酶（OST-PTP）是一种受体蛋白，其表达在成骨细胞分化过程中以及对骨重塑调节剂（如甲状旁腺激素和维生素D3）的反应中受到高度调控。为了探索该基因调控中重要的分子机制和信号通路，我们对小鼠OST-PTP cDNA和基因的结构组织进行了表征，并确定了其染色体定位。小鼠cDNA约为5.5 kb，包括5.1 kb的编码序列、315 bp的5'非翻译区和102 bp的3'非翻译区。它在第8天分化的MC3T3成骨细胞中表达为单一的约5.8 kb转录本。尽管与大鼠OST-PTP cDNA高度同源，但小鼠cDNA在5'非翻译区有一个74 bp的插入片段，其中包含几个潜在的转录因子结合位点，如AP-2和NFκB。小鼠OST-PTP（mOST-PTP）基因是一个单拷贝基因，包含35个外显子，跨度仅为20.65 kb。因此，它是已表征的受体PTP基因中最小的基因。这是由于缺乏大的内含子以及外显子的保守空间组织，这些外显子在mOST-PTP分子中编码特定的蛋白质基序。对推定的mOST-PTP启动子的序列分析揭示了基础元件以及许多与骨基因调控相关的潜在顺式作用调节元件。特别有趣的是在-1867位置发现的单个成骨细胞特异性元件，称为骨钙素特异性元件2（OSE2），以及在整个启动子和5'非翻译区发现的许多维生素D反应元件（VDRE）和NFκB位点。荧光原位杂交研究表明，mOST-PTP定位于小鼠染色体1的F-G区域，该区域与人染色体1q32-33区段同线。对mOST-PTP cDNA和基因的这种表征将有助于未来探索该磷酸酶在骨生成过程中调控机制的实验。
