Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation

Matrix metalloproteinase-13 (MMP-13) plays a critical role in parathyroid hormone (PTH)-induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP-13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8-bromo cyclic adenosine mono phosphate (8-Br-cAMP) treatment. We further demonstrate that in the presence of 8-Br-cAMP, the wild-type Runx2 construct stimulated MMP-13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP-13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling.     

Nmp4/CIZ对成骨细胞功能的调节作用

核基质蛋白4(nuclear matrix protein,Nmp4),亦称p130cas结合锌指蛋白(cas-interacting zinc finger protein,CIZ),是一种位于细胞核及粘附斑且具有核质穿梭功能的转录调控因子.体内实验证明,Nmp4/CIZ抑制骨形成活性进而抑制骨密度和骨量增加,而对骨吸收参数无显著影响.Nmp4/CIZ通过特异性结合成骨细胞Ⅰ型胶原α1链和基质金属蛋白酶启动子上游调控序列,调节其转录表达,促进骨转换.此外,Nmp4/CIZ抑制骨形态蛋白和甲状旁腺激素诱导的成骨细胞分化,抑制成骨细胞增殖并促进凋亡发生.Nmp4/CIZ参与调节成骨细胞力学-化学信号转导,其表达沉默可抑制尾悬吊诱导的小鼠骨量减少,并促进流体剪切力诱导的成骨细胞β-catenin信号途径.这些体内外实验证据表明,Nmp4/CIZ主要通过负调控机制发挥作用,提示这是一个潜在的骨丢失治疗药物靶点.     

Differential sequence dynamics of homopolymeric and alternating AT tracts in a small plasmid DNA

The location of OsO4 bispyridine hyper- and hyporeactivity in a small deletion derivative of plasmid ColE1 (PTC12, 1727 bp) has been determined for approximately 70% of the molecule. Thymine bases in homopolymeric (dA)n.(dT)n tracts (n greater than or equal to 4) were always found to be resistant toward OsO4 modification. DNA supercoiling did not destabilize these tracts. The extent of OsO4 bispyridine reactivity of homopolymeric (dA)n.(dT)n tracts, where n = 3, was found to be dependent on the rate of base unpairing of the sequence immediately 5' and 3' to the tract. Repressed OsO4 reactivity of thymine bases in (dA)3.(dT)3 tracts was observed if immediately both 5' and 3' to the tract were stable DNA sequences composed of GC base pairs and/or a homopolymeric (dA)n.(dT)n tract (n greater than or equal to 4). Homopolymeric tracts of n = 3 not having adjacent sequences with repressed unpairing rates did not show reduced levels of OsO4 bispyridine reactivity. Alternating d(TA)n tracts (n greater than or equal to 2) were found to exhibit hyperreactivity with OsO4. The extent of this hyperreactivity was dependent on the length of the tract and superhelical torsional stress. The distribution and frequency of homopolymeric (dA)n.(dT)n (n greater than or equal to 4) tracts in Escherichia coli promoter sequences were examined, and the possible implications of these tracts on promoter function are discussed.     

Endogenous FGF‐2 is critically important in PTH anabolic effects on bone

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2?/? mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2?/? osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2?/? osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2?/? osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2?/? osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2?/? osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2?/? osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2?/? osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2?/? mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009. © 2008 Wiley‐Liss, Inc.     

Local enrichment with homopolymeric (dA/dT) DNA in genomes of some lower dipterans and Drosophila melanogaster

An investigation into the chromosomal localization of homopolymeric dA/dT was carried out with species of the genera Rhynchosciara, Chironomus, Drosophila and several other taxa. In situ hybridisation probing mitotic and polytene chromosomes with RNA homopolymers was performed, followed by immunological detection of the DNA/RNA hybrid. Use of this method allowed us to assess specific regions of some dipteran genomes, where the signal was generally, but not always, located in heterochromatic regions. Human and Drosophila chromosome regions known to contain dA/dT runs of up to 153 bp were devoid of consistent labelling. The stability of the rA/dT hybrid formed in situ was in agreement with the T(m) for long rA/dT hybrid complexes, suggesting that the method used in this work is able to identify unusually long homopolymeric dA/dT tracts.