Melanocortin receptor accessory protein 2 (MRAP2) plays an important role in regulating melanocortin receptors. In zebrafish, MRAP2a and MRAP2b show distinct pharmacological effects on MC4R activity, but how MRAP2 protein regulates other zebrafish melanocortin receptors is barely studied. Zebrafish have two mc5r genes: mc5ra and mc5rb, it is still vague which one is the homologous isoform to the mammalian paralog. Here, we utilize synteny and phylogenetic analysis to demonstrate the evolutionary conservation of zebrafish MC5Ra and MC5Rb among different species. We also show that MRAP2a and MRAP2b could interact and regulate surface expression of two MC5R receptors. Bimolecular fluorescence complementation (BiFC) studies suggest that zebrafish MC5Rs could form homo- and heterodimers, which are suppressed by co-expression with MRAP2 proteins. In comparison with mammalian MC5R-MRAP2 system and different pharmacological effects of zMRAP2 protein on MC5Rs, zmc5ra is identified as the evolutionary homologous paralog to the mammals, and it is regulated by metabolic state in zebrafish brain region.
Ming Zhu, Meng Wang, Yijun Chen and Chao Zhang
Xiao-Hua Wang, Hao-Meng Wang, Bao-Lei Zhao, Peng Yu and Zhen-Chuan Fan
Melanocortin 4 receptor (MC4R) is a key factor in regulating energy homeostasis, and null mutations occurring in the gene encoding MC4R cause severe early-onset morbid obesity in humans. Many obesity-causing mutations affecting MC4R clinically identified so far lead to failure of mutant receptors to shuttle to the plasma membrane. In this study, we show that a novel human MC4R antagonist, Ipsen 17, acted as an pharmacological chaperone of human MCR4. As tested with 12 obesity-causing human MC4R variants including S58C, E61K, N62S, I69T, P78L, C84R, G98R, T162I, R165W, W174C, C271Y, and P299H, Ipsen 17 was found to be the most universal pharmacological chaperone of MC4R reported so far because it can completely rescue nearly all mutant receptors (except P299H) with the highest potency (an EC50 value of approximately 10−8 M) and efficiency when compared with results for other tested pharmacological chaperones of MC4R including ML00253764, PBA, MTHP, PPPone, MPCI, DCPMP, and NBP described in the literature. Once restored to the plasma membrane, defective human MC4R variants responded to α-MSH stimulation with an EC50 value of approximately 10−8 M and displayed dramatically enhanced signaling ability (except for G98R) in a mutant-specific efficacy and potency profile. Taken together, these results indicate that Ipsen 17 represents a candidate for the development of a targeted treatment of severe early-onset morbid obesity caused by a large subset of inherited mutations in the human MC4R gene.
Meng Ren, Qingbo Guan, Xia Zhong, Bendi Gong, Ying Sun, Wei Xin, Jun Guo, Hai Wang, Ling Gao and Jiajun Zhao
It is known that decreased apoptosis of thyrocytes may be involved in the formation of goiters in patients with Graves’ disease, and growth factors are involved in regulating the size of the thyroid gland. The purpose of our study was to investigate mRNA and protein levels of an antiapoptotic protein, namely, Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). The results showed that in FRTL thyroid cells, treatment with IGF-I upregulated mRNA and protein levels of FLIP in a dose-dependant manner. While a specific nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, blocked this effect. Further study demonstrated that IGF-I induced the DNA-binding activity of NF-κB in association with decreased expression of the NF-κB inhibitory protein IκBα . These findings implied that IGF-I increased FLIP expression by enhancing the activation of NF-κB in FRTL thyroid cells. Using a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we also found that PI3K was involved in the pathway by which IGF-I activated NF-κB and increased FLIP expression. When treated with IGF-I and LY294002, decreased NF-κB DNA binding activity and increased expression of IκBα protein were detected in cultured thyroid cells, which further confirmed that NF-κB was under the control of the PI3K pathway. Taken together, our results suggest that IGF-I regulates the expression of FLIP in FRTL cells by activating the PI3K/NF-κB cascade.
Ni Yan, Shuai Meng, Rong-Hua Song, Qiu Qin, Xuan Wang, Qiuming Yao, Yanfei Jiang, Wenjuan Jiang, Liangfeng Shi, Jian Xu and Jinan Zhang
Autoimmune thyroid disease (AITD) comprises Graves' disease (GD) and Hashimoto's thyroiditis (HT). IL37 has been recently proved to be a natural suppressor for innate immunity and acquired immunity. Therefore, this study was conducted to identify the association of IL37 genetic polymorphisms with AITD in Chinese Han population. Polymorphisms of rs3811046/rs3811047/rs2723176/rs272186 in the IL37 gene were assessed in a case–control study comprising 701 GD patients, 301 HT patients and 939 controls. Genetic variants were genotyped by multiplex polymerase chain reaction and ligase detection reaction. The frequencies of the minor allele A of rs2723176 and A of rs2723186 were significantly lower in the GD patients than in the controls (P=0.014, OR=0.774; P=0.014, OR=0.777). After gender stratification, the rs3811046 G allele and the rs3811047/rs2723186 A allele were both significantly associated with a decreased risk of GD in female patients (P=0.030, OR=0.777; P=0.023, OR=0.774; P=0.029, OR=0.761). However, none of the four single nucleotide polymorphisms of IL37 gene showed any significant association with HT. Moreover, haplotype analysis revealed the GCG haplotype conferred increased risk for GD as a whole and in female GD patients (OR=1.213; OR=1.320). The ACG haplotype was associated with an increased risk of HT as a whole (OR=1.567) and in male GD patients (OR=1.820). In contrast, the AAA haplotype showed a protective role for GD as a whole (OR=0.760) and in female GD patients (OR=0.765). Our study strongly supports that the IL37 gene variants are associated with the susceptibility to AITD.
Ming-Qing Li, Xiao-Fan Hou, Shi-Jian Lv, Yu-Han Meng, Xiao-Qiu Wang, Chuan-Ling Tang and Da-Jin Li
Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinβ1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17β-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2–CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinβ1 signal pathway.
Kamran Ullah, Tanzil Ur Rahman, Hai-Tao Pan, Meng-Xi Guo, Xin-Yan Dong, Juan Liu, Lu-Yang Jin, Yi Cheng, Zhang-Hong Ke, Jun Ren, Xian-Hua Lin, Xiao-Xiao Qiu, Ting-Ting Wang, He-Feng Huang and Jian-Zhong Sheng
Previous studies have shown that increasing estradiol concentrations had a toxic effect on the embryo and were deleterious to embryo adhesion. In this study, we evaluated the physiological impact of estradiol concentrations on endometrial cells to reveal that serum estradiol levels probably targeted the endometrium in controlled ovarian hyperstimulation (COH) protocols. An attachment model of human choriocarcinoma (JAr) cell spheroids to receptive-phase endometrial epithelial cells and Ishikawa cells treated with different estradiol (10−9 M or 10−7 M) concentrations was developed. Differentially expressed protein profiling of the Ishikawa cells was performed by proteomic analysis. Estradiol at 10−7 M demonstrated a high attachment rate of JAr spheroids to the endometrial cell monolayers. Using iTRAQ coupled with LC–MS/MS, we identified 45 differentially expressed proteins containing 43 significantly upregulated and 2 downregulated proteins in Ishikawa cells treated with 10−7 M estradiol. Differential expression of C3, plasminogen and kininogen-1 by Western blot confirmed the proteomic results. C3, plasminogen and kininogen-1 localization in human receptive endometrial luminal epithelium highlighted the key proteins as possible targets for endometrial receptivity and interception. Ingenuity pathway analysis of differentially expressed proteins exhibited a variety of signaling pathways, including LXR/RXR activation pathway and acute-phase response signaling and upstream regulators (TNF, IL6, Hmgn3 and miR-140-3p) associated with endometrial receptivity. The observed estrogenic effect on differential proteome dynamics in Ishikawa cells indicates that the human endometrium is the probable target for serum estradiol levels in COH cycles. The findings are also important for future functional studies with the identified proteins that may influence embryo implantation.