Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(−) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression of Rhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(−) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation of Rhob in RAW-GR(−) and RAW264.7 cells by transfection with wild-type Rhob expression vector (Rhob-wt) or constitutively activated Rhob plasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression of Rhob by RNA interference technique or the activity of Rhob by transfection with dominant negative Rhob mutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(−) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate that Rhob plays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR→Akt→Rhob signaling and Rhob negatively regulates the proliferation of RAW264.7 cells.
Xiaohui Wang, Yuxia Chen, Yan Wang, Xiaoyan Zhu, Yuanyuan Ma, Shimin Zhang and Jian Lu
Shih-Lu Wu, Tin-Yun Ho, Ji-An Liang and Chien-Yun Hsiang
The sodium/iodide symporter (SLC5A5; also known as NIS), a transmembrane glycoprotein principally in the thyroid gland, is responsible for the accumulation of iodide necessary for thyroid hormones. Our previous study indicated that a novel exon 6 deletion (residues 233–280) in SLC5A5 loses the iodide uptake activity. Herein we characterized the role of His-226 in iodide transport of SLC5A5. His-226, a highly conserved extracellular residue among SLC5A5 homologs, was replaced with alanine, aspartic acid, glutamic acid, or lysine. All the SLC5A5 mutants were expressed normally in the cells and targeted correctly to the plasma membrane. However, all of the mutants displayed severe defects in iodide uptake, suggesting that His-226 was critical for iodide uptake. Kinetic analysis further showed that mutation at His-226 led to a dramatic decrease in V max. These findings suggested that the decreased levels of iodide uptake activity of SLC5A5 mutants resulted from lower catalytic rates. In conclusion, our data first identified the involvement of extracellular charged amino acid residue in the iodide uptake ability of SLC5A5.
Te Du, Liu Yang, Xu Xu, Xiaofan Shi, Xin Xu, Jian Lu, Jianlu Lv, Xi Huang, Jing Chen, Heyao Wang, Jiming Ye, Lihong Hu and Xu Shen
Vincamine, a monoterpenoid indole alkaloid extracted from the Madagascar periwinkle, is clinically used for the treatment of cardio-cerebrovascular diseases, while also treated as a dietary supplement with nootropic function. Given the neuronal protection of vincamine and the potency of β-cell amelioration in treating type 2 diabetes mellitus (T2DM), we investigated the potential of vincamine in protecting β-cells and ameliorating glucose homeostasis in vitro and in vivo. Interestingly, we found that vincamine could protect INS-832/13 cells function by regulating G-protein-coupled receptor 40 (GPR40)/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathway, while increasing glucose-stimulated insulin secretion (GSIS) by modulating GPR40/cAMP/Ca2+/CaMKII pathway, which reveals a novel mechanism underlying GPR40-mediated cell protection and GSIS in INS-832/13 cells. Moreover, administration of vincamine effectively ameliorated glucose homeostasis in either HFD/STZ or db/db type 2 diabetic mice. To our knowledge, our current work might be the first report on vincamine targeting GPR40 and its potential in the treatment of T2DM.
Laura E Pascal, Khalid Z Masoodi, June Liu, Xiaonan Qiu, Qiong Song, Yujuan Wang, Yachen Zang, Tiejun Yang, Yao Wang, Lora H Rigatti, Uma Chandran, Leandro M Colli, Ricardo Z N Vencio, Yi Lu, Jian Zhang and Zhou Wang
Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivo. Ell2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.