The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protein (GFP)-tagged receptors were found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRNRCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.
Z Yu, CH Lee, C Chinpaisal and LN Wei
Fu-Qing Yu, Chun-Sheng Han, Wei Yang, Xuan Jin, Zhao-Yuan Hu and Yi-Xun Liu
In the present study, we started out to test whether the follicle-stimulating hormone (FSH)-activated p38 MAPK signaling cascade was involved in the regulation of steroidogenesis in granulosa cells (GCs). GCs were prepared from the ovaries of DES-treated immature rats and cultured in serum-free medium. Treatment of GCs with FSH (50 ng/ml) induced the phosphorylation of p38 MAPK rapidly with the phosphorylation being observed within 5 min and reaching the highest level at 30 min. Such activation was protein kinase A-dependent as indicated by the results using specific inhibitors. FSH stimulated the production of progesterone and estradiol as well as the expression of the steroidogenic acute regulatory protein (StAR) in a time-dependent manner, with a maximum level being observed in the production of progesterone and StAR at 48 h. Moreover, the potent p38 MAPK inhibitor SB203580 (20 μM) augmented FSH-induced progesterone and StAR production, while reduced FSH-induced estradiol production at the same time (P<0.01). RT-PCR data showed that inclusion of SB203580 in the media enhanced FSH-stimulated StAR mRNA production, while decreased the FSH-stimulated P450arom mRNA expression (P<0.05). Immunocytochemical studies showed that FSH treatment together with the inhibition of p38 MAPK activity resulted in a higher expression of StAR in mitochondria than FSH treatment alone. FSH also significantly up-regulated the protein level of LRH-1, a member of the orphan receptor family that activates the expression of P450arom in ovaries and testes. p38 MAPK inactivation down-regulated the basal and FSH-induced LRH-1 expression significantly. The intra-cellular level of DAX-1, another orphan receptor that inhibits StAR expression, also decreased upon p38 MAPK being inactivated. For the first time, the present study suggests that FSH-activated p38 MAPK signal pathway regulates progesterone and estrogen production in GCs differentially, and that the transcription factors LRH-1 and DAX-1 might play important roles in the process.
Wenjing Wu, Jiayao Fu, Yijing Gu, Yu Wei, Pengfei Ma and Junhua Wu
Emerging evidence has indicated that estrogen deficiency contributes to osteoporosis by affecting the level of inflammation. The inflammation microenvironment affects many cellular physiological processes, one of which may be cellular senescence according to previous studies. Senescent cells cannot function normally and secrete inflammatory cytokines and degradative proteins, which are referred to as senescence-associated secretory phenotype (SASP) factors, inducing further senescence and inflammation. Thus, stopping this vicious cycle may be helpful for postmenopausal osteoporosis treatment. Here, we used ovariectomized (OVX) mice as an estrogen-deficient model and confirmed that OVX bone marrow mesenchymal stem cells (BMSCs) displayed a senescent phenotype and upregulated SASP factor secretion both in vitro and in vivo. Furthermore, JAK2/STAT3, an important cytokine secretion-related signaling pathway that is associated with SASP secretion, was activated. Estrogen addition and estrogen receptor blockade confirmed that the JAK2/STAT3 axis participated in OVX BMSC senescence by mediating SASP factors. And JAK inhibition reduced SASP factor expression, alleviated senescence and enhanced osteogenic differentiation. Intraperitoneal injection of a JAK inhibitor, ruxolitinib, prevented bone loss in OVX mice. Collectively, our results revealed that JAK2/STAT3 plays an important role in the inflammation-senescence-SASP feedback loop in OVX BMSCs and that JAK inhibition could be a new method for treating postmenopausal osteoporosis.
Xuanchun Wang, Wei Gong, Yu Liu, Zhihong Yang, Wenbai Zhou, Mei Wang, Zhen Yang, Jie Wen and Renming Hu
We report the identification of a novel secreted peptide, INM02. The mRNA transcript of human INM02 gene is about 3.0 kb. Its open-reading frame contains 762 bps and encodes a protein of 254 amino acids. Northern blot analysis demonstrates that INM02 mRNA is widely expressed in rat tissues, especially with abundant quantities in pancreatic islets, testis, and bladder tissue. We have expressed recombinant INM02 protein and generated rabbit anti-INM02 polyclonal antibodies. We show here that INM02 could be detectable in human serum by ELISA. We also present evidence that INM02 mRNA expression could be regulated by glucose. Experiments on both MIN6 cells and intact isolated islets demonstrate that INM02 mRNA levels are increased more than threefold by high glucose (25 mM) when compared with low glucose (5.5 mM). ELISA analysis shows that secretion of INM02 is significantly augmented by high glucose in vitro. It is speculated that as a novel secreted protein, INM02 is associated with functions of pancreatic islets, especially of β-cells.
Hong-Hui Wang, Qian Cui, Teng Zhang, Lei Guo, Ming-Zhe Dong, Yi Hou, Zhen-Bo Wang, Wei Shen, Jun-Yu Ma and Qing-Yuan Sun
As a fat storage organ, adipose tissue is distributed widely all over the body and is important for energy supply, body temperature maintenance, organ protection, immune regulation and so on. In humans, both underweight and overweight women find it hard to become pregnant, which suggests that appropriate fat storage can guarantee the female reproductive capacity. In fact, a large mass of adipose tissue distributes around the reproductive system both in the male and female. However, the functions of ovary fat pad (the nearest adipose tissue to ovary) are not known. In our study, we found that the ovary fat pad-removed female mice showed decreased fertility and less ovulated mature eggs. We further identified that only a small proportion of follicles developed to antral follicle, and many follicles were blocked at the secondary follicle stage. The overall secretion levels of estrogen and FSH were lower in the whole estrus cycle (especially at proestrus); however, the LH level was higher in ovary fat pad-removed mice than that in control groups. Moreover, the estrus cycle of ovary fat pad-removed mice showed significant disorder. Besides, the expression of FSH receptor decreased, but the LH receptor increased in ovary fat pad-removed mice. These results suggest that ovary fat pad is important for mouse reproduction.
Jiashu Yu, Zhongyan Shan, Wei Chong, Jinyuan Mao, Yuxiu Geng, Caixia Zhang, Qian Xing, Weiwei Wang, Ningna Li, Chenling Fan, Hong Wang, Hongmei Zhang and Weiping Teng
Acute and excessive iodine supplementation leads to iodine-induced thyroid cytotoxicity. Excessive oxidative stress has been suggested to be one of the underlying mechanisms in the development of thyroid cytotoxicity. The aim of this study was to investigate whether vitamin E (VE), an important antioxidant, could ameliorate iodine-induced thyroid cytotoxicity. A goiter was induced in rats by feeding a low-iodine (LI) diet for 12 weeks. Involution of hyperplasia was obtained by administering a twofold physiological dose of iodine in feeding water with/without the supplementation of 25-, 50-, or 100-fold physiological dose of VE in the LI diet for 4 weeks. In iodine-supplemented rats, thyroid epithelial cell ultrastructure injuries remained and were more severe. Relative weights of iodine-induced involuting glands were significantly reduced compared with the goiter, but still higher than control. Immunohistochemistry indicated that the expression of 4-hydroxynonenal, 8-hydroxyguanine, peroxiredoxin 5, and CD68 in thyroid increased (P<0.01), whereas thioredoxin reductase 1 decreased (P<0.01). VE supplementation attenuated thyroid cytotoxicity induced by iodine. A 50-fold VE dose was optimal in attenuating twofold iodine-induced thyroid cytotoxicity. However, VE supplementation did not reduce the weight or relative weight of the iodine-induced involuting gland. These results show that excess iodine leads to thyroid damage and VE supplementation can partly ameliorate iodine-induced thyroid cytotoxicity.
Yan-Hong Bu, Yu-Ling He, Hou-De Zhou, Wei Liu, Dan Peng, Ai-Guo Tang, Ling-Li Tang, Hui Xie, Qiu-Xia Huang, Xiang-Hang Luo and Er-Yuan Liao
Insulin receptor substrate 1 (IRS1) is an essential molecule for the intracellular signaling of IGF1 and insulin, which are potent anabolic regulators of bone metabolism. Osteoblastic IRS1 is essential for maintaining bone turnover; however, the mechanism underlying this regulation remains unclear. To clarify the role of IRS1 in bone metabolism, we employed RNA interference to inhibit IRS1 gene expression and observed the effects of silencing this gene on the proliferation and differentiation of and the expression of matrix metallopeptidase (MMP) and tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) in MC3T3-E1 cells. Our results showed that IRS1 short hairpin RNAs can effectively suppress the expression of IRS1, and inhibit the phosphorylation of AKT in IRS1 pathway; reduce the expression of MMP2, MMP3, MMP13, and MMP14, decrease the expression of TNFRSF11B and RANKL (also known as tumor necrosis factor (ligand) superfamily, member 11), however increase the RANKL/TNFRSF11B ratio; decrease cell survival, proliferation, and mineralization, and impair the differentiation of MC3T3-E1 cells. The downregulation of IRS1 had no effect on the expression of MMP1. Our findings suggest that IRS1 not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover.