甲状旁腺素、胰岛素样生长因子-1可加速成骨样细胞ROS17/2.8增值周期的进程

甲状旁腺素(parathyroid hormone,PTH)不仅在调节钙磷代谢中可促进骨发生破骨细胞性溶骨,也可促进骨的合成代谢作用。近年发现PTH还可促进成骨细胞的增殖分化。其细胞生物学和分子生物学机理尚待研究。本实验以成骨样细胞ROS17/2.8为研究材料,以胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)为细胞增殖的阳性对照,检测了PTH对DNA合成、细胞周期进程及cyclin E和cyclinA的表达,以求探讨PTH促成骨细胞增殖时对细胞周期的影响。结果表明,PTH可促进DNA合成,改变细胞周期各时相细胞比例及增加cyclin E和cyclin A的表达。该结果提示PTH可加速成骨细胞增殖周期的进程。     

甲状旁腺素和胰岛素样生长因子促成骨样细胞ROS 17/2.8增殖分化中的协同作用

 间歇性小剂量地给予甲状旁腺素 (parathyroid hormone,PTH)可促进成骨 .胰岛素样生长因子 - I(insulin- like growth factor- I,IGF- I)由成骨细胞所产生并贮存于骨基质中 ,可促进成骨细胞的增殖分化 .为进一步了解向钙性激素和骨源性生长因子对骨生长的影响 ,利用成骨样细胞 ROS1 7/ 2 .8进行体外实验 ,观察了 PTH和 IGF- I这两种在骨生长和代谢中有重要作用的激素和因子相互作用的效果 ,并对其相互作用机制作出初步探讨 .结果显示 :联合使用 IGF- I及 PTH(间歇性给药 )时 ,(1 ) SRB(sodium rhodamine B,SRB)染色显示经 PTH(1 0 -9mol/ L,间歇给药 )和 IGF- I(1 0 -9mol/ L)联合处理的细胞 ,其数目明显增加 ,且明显高于单独处理组 ;(2 ) 3H- Td R参入增加 ,也明显高于单独处理组 ;(3)与增殖相关的原癌基因 (c- fos,c- jun,c- ki- ras)的表达增强 ,明显高于单独处理组 ;(4)骨钙素 (osteocalcin)基因 m RNA表达增强 ,明显高于单独处理组 ;(5) IGF- I(1 0 -8mol/L,1 0 -9mol/ L)可使 PTH受体基因 m RNA表达增强 .这些结果提示 PTH和 IGF- I在成骨样细胞ROS 1 7/ 2 .8增殖分化中具有协同作用 ,原癌基因的表达增强可能是其作用的一个环节 .此外 ,IGF- I可能通过增强 PTH受体表达 ,使细胞对 PTH的反应性增强     

肿瘤坏死因子α对ROS17/2.8细胞甲状旁腺素受体的下行调节

肿瘤坏死因子α(TNFα)是激活的单核巨噬细胞分泌的蛋白质,分子量17kD。其多功能性和选择性抑制肿瘤细胞生长的作用受到高度重视。我们的实验表明:TNFα(3×10~(-10)-1×10~(-7)mol/L)能显著降低大鼠成骨肉瘤细胞株ROS17/2.8的甲状旁腺素(PTH)受体总结合率,比对照降低7.47-37.45％,且与TNFα的浓度呈正相关。时间曲线显示,TNFα作用时间越长,受体总结合率降低越明显。Scatchard作图表明PTH受体数目降低而其亲和力无显著变化。细胞周期分析显示,TNFα(3.83×10~(-10) mol/L作用3天)能抑制S期DNA合成。可见TNFα通过减少PTH受体数目以调节骨代谢。同时通过抑制DNA的合成以调节骨细胞的增殖。     

甲状腺素及维甲酸对成骨样细胞甲状旁腺素受体的调节作用

研究了甲状腺素（T3／T4）及维甲酸（RA）对大鼠成骨样细胞ROS17／2．8细胞林甲状旁腺素（PTH）受体的调节作用．实验结果表明：细胞经T3／T4处理后,可显著增高PTH受体结合率及碱性磷酸酶活性,以及PTH受体mRNA的表达．细胞经RA处理后,则相反地降低PTH受体结合率及碱性磷酸酶活性．     

甲状旁腺素对成骨样细胞增殖的调节作用

甲状旁腺素（ＰＴＨ）是调节钙磷代谢的经典激素,有报道ＰＴＨ对其靶细胞－成骨细胞有促增殖分化作用。经多层次、多水平的实验研究证实,ＰＴＨ对成骨样细胞ＲＯＳ１７／２．８确有促增殖作用。（１）细胞计数、ＭＴＴ［３－（４,５－ｄｉｍｅｔｈｙｌｔｈｉａ－ｚｏｌ－ｚ－ｙｉ）２,５－ｄｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍｂｒｏｍｉｄｅ］测定及ＳＲＢ（ｓｏｄｉｕｍｒｈｏｄａｍｉｎｅＢ,ＳＲＢ）染色均显示经ＰＴＨ（１０－９ｍｏｌ／Ｌ）处理的细胞,其数目明显增加;（２）３Ｈ－ＴｄＲ参入增加;（３）与增殖相关的原癌基因（ｃ－ｆｏｓ、ｃ－ｊｕｎ、ｃ－ｋｉ－ｒａｓ和ｃ－ｍｙｃ）的表达增强;（４）成骨细胞特征性蛋白－碱性磷酸酶活性降低．这些结果不仅表明该激素具有非经典样作用,同时意味着激素也参与其靶细胞增殖分化的调节作用     

核苷对ROS17/2.8细胞株腺苷酸环化酶活性的影响

 大鼠成骨肉瘤细胞株(ROS17/2.8)系甲状旁腺素(PTH)的靶细胞。当该细胞质膜上的PTH受体与PTH结合后,可激发腺苷酸环化酶(AC)的活性。腺苷及四种不同的核苷酸(AMP、GMP、UMP、CMP)单独对AC无明显效应,但却可抑制PTH对AC的刺激作用。而鸟苷或木糖腺苷则可显著增强PTH对AC的刺激作用。提示核苷的不同代谢物在代谢调节中的多样化作用。     

肿瘤坏死因子α对大鼠成骨样细胞增殖与甲状旁腺素受体表达的影响

大鼠成骨样细胞ＲＯＳ１７／２．８经肿瘤坏死因子α处理后,发现其ｃ－ｍｙｃ,ｃ－ｆｏｓ,ｃ－ｊｕｎ等原癌基因ｍＲＮＡ的表达降低,细胞增殖明显受到抑制;同时,其ＰＴＨ受体的表达以及ＰＴＨ经受体刺激后引起的细胞内ｃＡＭＰ的增加也受到抑制。提示ＴＮＦα可抑制大鼠在骨要细胞ＲＥＯＳ１７／２．８的增殖和分化。     

肿瘤坏死因子α对大鼠成骨样细胞增殖与甲状旁腺素受体表达的影响

大鼠成骨样细胞ROS17／2．8经肿瘤坏死因子α处理后,发现其c－myc,c－fos,c－jun等原癌基因mRNA的表达降低,细胞增殖明显受到抑制;同时,其PTH受体的表达以及PTH受体经刺激后引起的胞内cAMP的增加也受到抑制．提示TNFα可抑制大鼠成骨样细胞ROS17／2．8的增殖和分化．     

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

Activation and Multiple-Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.     

Physiologic Levels of β-Amyloid Activate Phosphatidylinositol 3-Kinase with the Involvement of Tyrosine Phosphorylation

Abstract: The β-amyloid protein (Aβ) peptide plays an important role in Alzheimer's disease, but the potential actions of physiologic levels of Aβ (225–625 p M ) have not been explored. We recently showed that picomolar doses of Aβ can stimulate tyrosine phosphorylation of neuronal cells and now show that leads to the activation of the lipid kinase phosphatidylinositol 3-kinase (PI3 kinase). Three independent lines of evidence support the hypothesis that Aβ is activating PI3 kinase through a tyrosine kinase-mediated mechanism. Immunoblotting studies show that Aβ induces tyrosine phosphorylation of p85 as well as association of the p85 subunit of PI3 kinase with tyrosine-phosphorylated proteins. Studies of membrane proteins show that Aβ induces a translocation of p85 to membrane-bound glycoproteins, which are likely to be receptors. Finally, direct studies of PI3 kinase activity in both anti-phosphotyrosine immunocomplexes and wheat germ agglutinin precipitates show that Aβ increases formation of the product of PI3 kinase. Wortmannin, a selective inhibitor of PI3 kinase, blocks this Aβ-stimulated PI3 kinase activity. Thus, physiologic levels of Aβ stimulate tyrosine phosphorylation and PI3 kinase activity.     

Inhibition of receptor internalization attenuates the TNFalpha-induced ROS generation in non-phagocytic cells

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNFalpha induces ROS generation are not well characterized. Here, we report that TNFalpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNFalpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNFalpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNFalpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes

Ras can become activated via multiple distinct receptors in T lymphocytes. However, mechanistic studies of Ras signaling in normal T cells have been hampered by the lack of an efficient technology for gene transfer into resting post-thymic cells. We have overcome this limitation by utilizing adenoviral transduction of T cells from Coxsackie/adenovirus receptor transgenic mice. Unexpectedly, dominant negative Ras17N blocked activation of Ras and ERK in response to IL-2R engagement but not TCR/CD3 ligation. However, TCR-induced ERK activation was suppressed by inhibitors of PKC and PLC-gamma. This first biochemical study of DN Ras in normal quiescent T cells reveals a striking contrast in Ras signaling via two receptors, and suggests that the principal mechanism of TCR-induced Ras activation in normal T cells may be distinct from that utilized in T-lineage tumor cell lines.     

The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf

Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.