The extracellular calcium-sensing receptor (CaR; alternate gene names, CaR or Casr) is a membrane-spanning G protein–coupled receptor. CaR is highly expressed in the parathyroid gland, and is activated by extracellular calcium (Ca2+o). Mice homozygous for null mutations in the CaR gene (CaR–/–) die shortly after birth because of the effects of severe hyperparathyroidism and hypercalcemia. A wide variety of functions have been attributed to CaR. However, the lethal CaR-deficient phenotype has made it difficult to dissect the direct effect of CaR deficiency from the secondary effects of hyperparathyroidism and hypercalcemia. We therefore generated parathyroid hormone–deficient (PTH-deficient) CaR–/– mice (Pth–/–CaR–/–) by intercrossing mice heterozygous for the null CaR allele with mice heterozygous for a null Pth allele. We show that genetic ablation of PTH is sufficient to rescue the lethal CaR–/– phenotype. Pth–/–CaR–/– mice survive to adulthood with no obvious difference in size or appearance relative to control Pth–/– littermates. Histologic examination of most organs did not reveal abnormalities. These Pth–/–CaR–/– mice exhibit a much wider range of values for serum calcium and renal excretion of calcium than we observe in control littermates, despite the absence of any circulating PTH. Thus, CaR is necessary for the fine regulation of serum calcium levels and renal calcium excretion independent of its effect on PTH secretion.
Claudine H. Kos, Andrew C. Karaplis, Ji-Bin Peng, Matthias A. Hediger, David Goltzman, Khalid S. Mohammad, Theresa A. Guise, Martin R. Pollak
While mast cells and basophils constitutively express the high-affinity IgE receptor (FcεRI), it is absent or weakly expressed on APCs from normal donors. FcεRI is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases. Despite its clinical relevance, data about FcεRI regulation on APCs are scarce. We show that in all donors intracellular α chain of the FcεRI (FcεRIα) accumulates during DC differentiation from monocytes. However, expression of γ chains of the FcεRI (FcεRIγ), mandatory for surface expression, is downregulated. It is low or negative in DCs from normal donors lacking surface FcεRI (FcεRIneg DCs). In contrast, DCs from atopics express surface FcεRI (FcεRIpos DCs) and show significant FcεRIγ expression, which can be coprecipitated with FcεRIα. In FcεRIneg DCs lacking FcεRIγ, immature and core glycosylated FcεRIα accumulates in the endoplasmic reticulum. In FcεRIpos DCs expressing FcεRIγ, an additional mature form of FcεRIα exhibiting complex glycosylation colocalizes with FcεRIγ in the Golgi compartment. IgE binding sustains surface-expressed FcεRI on DCs from atopic donors dependent on baseline protein synthesis and transport and enhances their IgE-dependent APC function. We propose that enhanced FcεRI on DCs from atopic donors is driven by enhanced expression of otherwise limiting amounts of FcεRIγ and is preserved by increased IgE levels.
Natalija Novak, Carmen Tepel, Susanne Koch, Klaudia Brix, Thomas Bieber, Stefan Kraft
We performed a genetic and epigenetic study of the hMLH1 and hMSH2 mismatch repair genes in resected primary tumors from 77 non-small cell lung cancer (NSCLC) patients. The molecular alterations examined included the loss of mRNA and protein expression as well as promoter methylation, and the allelic imbalance of the chromosomal regions that harbor the genes. We found that 78% and 26% of patients showed at least one type of molecular alteration within the hMLH1 and hMSH2 genes, respectively. Promoter methylation of the hMLH1 gene was present in 55.8% of tumors, and was significantly associated with the reduction in mRNA and protein expression (P = 0.001). A 72% concordance of aberrant methylation in sputum samples with matched resected tumors was found. In addition, a 93% consistency between the promoter methylation and the mRNA expression of the hMSH2 gene was found in 14 female NSCLC patients. However, no correlation was found between the expression of hMLH1 and hMSH2 proteins and the allelic imbalance of five microsatellite markers closely linked to the genes. Our results suggest that hMLH1 is the major altered mismatch repair gene involved in NSCLC tumorigenesis, and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMLH1 gene may be identified in sputum samples to serve as a potential diagnostic marker of NSCLC.
Yi-Ching Wang, Yung-Pin Lu, Ruo-Chia Tseng, Ruo-Kai Lin, Jer-Wei Chang, Jung-Ta Chen, Chuen-Ming Shih, Chih-Yi Chen
Although lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. Recently, it has been reported that overexpression of VEGF-C correlates with increased lymphatic vessel growth (lymphangiogenesis). However, the effect of VEGF-C–induced lymphangiogenesis on lymphedema has yet to be demonstrated. Here we investigated the impact of local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) on two animal models of lymphedema: one in the rabbit ear and the other in the mouse tail. In a rabbit model, following local phVEGF-C gene transfer, VEGFR-3 expression was significantly increased. This gene transfer led to a decrease in thickness and volume of lymphedema, improvement of lymphatic function demonstrated by serial lymphoscintigraphy, and finally, attenuation of the fibrofatty changes of the skin, the final consequences of lymphedema. The favorable effect of phVEGF-C on lymphedema was reconfirmed in a mouse tail model. Immunohistochemical analysis using lymphatic-specific markers: VEGFR-3, lymphatic endothelial hyaluronan receptor-1, together with the proliferation marker Ki-67 Ab revealed that phVEGF-C transfection potently induced new lymphatic vessel growth. This study, we believe for the first time, documents that gene transfer of phVEGF-C resolves lymphedema through direct augmentation of lymphangiogenesis. This novel therapeutic strategy may merit clinical investigation in patients with lymphedema.
Young-sup Yoon, Toshinori Murayama, Edwin Gravereaux, Tengiz Tkebuchava, Marcy Silver, Cynthia Curry, Andrea Wecker, Rudolf Kirchmair, Chun Song Hu, Marianne Kearney, Alan Ashare, David G. Jackson, Hajime Kubo, Jeffrey M. Isner, Douglas W. Losordo
The availability of mice lacking the mitochondrial uncoupling protein UCP1, has provided an opportunity to analyze the relationship between the capacity for energy expenditure and the development of obesity in response to a high-fat, high-sucrose diet. Congenic UCP1-deficient mice on a C57BL/6J genetic background show a temperature-dependent resistance to diet-induced obesity when compared with wild-type mice. This resistance, which occurs at 20°C, is quickly reversed when the ambient temperature is increased to 27°C. At 20°C, total oxygen consumption and physical activity of mutant and wild-type mice are indistinguishable; however, body temperature is higher in UCP1-deficient mice by 0.1–0.3°C, and respiratory quotient is slightly reduced. A reduced respiratory quotient, together with elevated β-hydroxybutyrate and reduced plasma fatty acid levels, suggests that the mutants oxidize a greater proportion of fat than wild-type mice, and that this possibly accounts for the resistance to diet-induced obesity. Although shivering is one alternative mechanism of thermogenesis that is probably used in UCP1-deficient mice, whether there are others remains to be determined. Nevertheless, our study underscores the paradox that elimination of the major thermogenic mechanism in the animal reduces rather than increases metabolic efficiency. We propose that in the absence of nonshivering thermogenesis, alternative, calorically more costly pathways of metabolism must be used to maintain body temperature.
Xiaotuan Liu, Martin Rossmeisl, Jennifer McClaine, Leslie P. Kozak
IGF-II is a mitogenic peptide that has been implicated in hepatocellular oncogenesis. Since the silencing of gene expression is frequently associated with cytosine methylation at cytosine-guanine (CpG) dinucleotides, we designed a methylated oligonucleotide (MON1) complementary to a region encompassing IGF2 promoter P4 in an attempt to induce DNA methylation at that locus and diminish IGF2 mRNA levels. MON1 specifically inhibited IGF2 mRNA accumulation in vitro, whereas an oligonucleotide (ON1) with the same sequence but with nonmethylated cytosines had no effect on IGF2 mRNA abundance. MON1 treatment led to the specific induction of de novo DNA methylation in the region of IGF2 promoter hP4. Cells from a human hepatocellular carcinoma (HCC) cell line, Hep 3B, were implanted into the livers of nude mice, resulting in the growth of large tumors. Animals treated with MON1 had markedly prolonged survival as compared with those animals treated with saline or a truncated methylated oligonucleotide that did not alter IGF2 mRNA levels in vitro. This study demonstrates that a methylated sense oligonucleotide can be used to induce epigenetic changes in the IGF2 gene and that inhibition of IGF2 mRNA accumulation may lead to enhanced survival in a model of HCC.
Xiaoming Yao, Ji-Fan Hu, Mark Daniels, Hadas Shiran, Xiangjun Zhou, Huifan Yan, Hongqi Lu, Zhilan Zeng, Qingxue Wang, Tao Li, Andrew R. Hoffman
Christopher S. Rogers, Carlos G. Vanoye, Bruce A. Sullenger, Alfred L. George Jr.
Hiroyasu Tsukaguchi, Akulapalli Sudhakar, Tu Cam Le, Trang Nguyen, Jun Yao, Joshua A. Schwimmer, Asher D. Schachter, Esteban Poch, Patricia F. Abreu, Gerald B. Appel, Aparecido B. Pereira, Raghu Kalluri, Martin R. Pollak
Loan K. Phan, Feng Lin, Charles A. LeDuc, Wendy K. Chung, Rudolph L. Leibel
Xinli Zhang, Shun’ichi Kuroda, Dale Carpenter, Ichiro Nishimura, Chia Soo, Rex Moats, Keisuke Iida, Eric Wisner, Fei-Ya Hu, Steve Miao, Steve Beanes, Catherine Dang, Heleni Vastardis, Michael Longaker, Katsuyuki Tanizawa, Norihiro Kanayama, Naoaki Saito, Kang Ting