Fu Qiang, Manolagas Stavros C, O'Brien Charles A
University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 587, Little Rock, AR 72205, USA.
Mol Cell Biol. 2006 Sep;26(17):6453-68. doi: 10.1128/MCB.00356-06.
RANKL, a protein essential for osteoclast development and survival, is stimulated by parathyroid hormone (PTH) via a PTH receptor 1/cyclic AMP (cAMP)/protein kinase A (PKA)/CREB cascade, exclusively in osteoblastic cells. We report that a bacterial artificial chromosome-based transcriptional reporter construct containing 120 kb of RANKL 5'-flanking region was stimulated by dibutyryl-cAMP in stromal/osteoblastic cells, but not other cell types. Full cAMP responsiveness was dependent upon a conserved 715-bp region located 76 kb upstream from the transcription start site, which we identified by sequential deletion analysis and by comparison of human and mouse genomic sequences in silico. This region contained conserved consensus sequences which bound CREB and the osteoblast-specific transcription factor Runx2, and when mutated blunted cAMP responsiveness. Overexpression of Runx2 potentiated cAMP responsiveness of the endogenous RANKL gene in a cell-type-specific manner. Lastly, PTH responsiveness of the endogenous RANKL gene was abrogated in mice from which we deleted this conserved upstream region. Thus, PTH responsiveness of the RANKL gene is determined by a distant regulatory region that responds to cAMP in a cell-type-specific manner and Runx2 may contribute to such cell-type specificity.
核因子κB受体活化因子配体（RANKL）是破骨细胞发育和存活所必需的一种蛋白质，甲状旁腺激素（PTH）通过PTH受体1/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）/CREB级联反应，仅在成骨细胞中刺激RANKL。我们报告，一个基于细菌人工染色体的转录报告构建体，包含120 kb的RANKL 5'侧翼区域，在基质/成骨细胞中被二丁酰环磷腺苷（dibutyryl-cAMP）刺激，但在其他细胞类型中未被刺激。完整的cAMP反应性依赖于位于转录起始位点上游76 kb处的一个保守的715 bp区域，我们通过序列缺失分析以及通过在计算机上比较人和小鼠的基因组序列确定了该区域。该区域包含与CREB和成骨细胞特异性转录因子Runx2结合的保守共有序列，当发生突变时会减弱cAMP反应性。Runx2的过表达以细胞类型特异性方式增强了内源性RANKL基因的cAMP反应性。最后，在我们删除了这个保守上游区域的小鼠中，内源性RANKL基因的PTH反应性被消除。因此，RANKL基因的PTH反应性由一个远距离调控区域决定，该区域以细胞类型特异性方式对cAMP作出反应，并且Runx2可能有助于这种细胞类型特异性。
