Myeloid Differentiation Factor 88 (MyD88)-Deficiency Increases Risk of Diabetes in Mice

Background

Multiple lines of evidence suggest innate immune response pathways to be involved in the development of obesity-associated diabetes although the molecular mechanism underling the disease is unknown. Recent observations suggest that saturated fatty acids can act as a ligand for toll-like receptor (TLR) 4, which is thought to mediate obesity-associated insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adapter protein for TLR/IL-1 receptor signaling, which is involved in the activation of inflammatory pathways. To evaluate molecular mechanisms linking obesity-associated diabetes down-stream of TLR4, we investigated physiological role of MyD88 in high-fat diet (HFD)-induced obesity.

Methodology/Principal Findings

In the present study, we found MyD88-deficient mice fed a HFD had increased circulating levels of insulin, leptin and cholesterol, as well as liver dysfunction (increased induction of ALT levels, increased activation of JNK and cleavage of PARP), which were linked to the onset of severe diabetes. On the other hand, TNF-α would not be involved in HFD-induced diabetes in MyD88-deficient mice, because TNF-α level was attenuated in MyD88-deficient mice fed with HFD.

Conclusions/Significance

The present finding of an unexpected role for MyD88 in preventing diabetes may provide a potential novel target/strategy for treating metabolic syndrome.     

Identification and Function of Myeloid Differentiation Factor 88 (MyD88) in Litopenaeus vannamei

Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.     

猪髓样分化因子MyD88特异性shRNA干扰载体的构建筛选及干扰效果评价

髓样分化因子My D88(myeloid differentiation factor 88)信号通路是一个具有多种调节功能的传导通路,在免疫反应、炎症反应及肿瘤的发生和发展过程中均发挥重要作用。构建猪(Sus scrofa)My D88基因的shRNA干扰载体,并在转录水平和蛋白质表达水平对其干扰效果进行验证,以筛选出干扰效果最优的干扰载体。根据猪My D88基因(Gen Bank登录号:KC766424.1)全长c DNA序列,利用Invitrogen公司在线设计软件设计出4对shRNA干扰序列,退火成双链后,分别将其插入到p Yr-1.1载体中,构建My D88基因的shRNA真核表达载体p Yr-1.1-pig My D88-sh1、p Yr-1.1-pig My D88-sh2、p Yr-1.1-pig My D88-sh3、p Yr-1.1-pig My D88-sh4,并通过双酶切和测序对其进行鉴定。构建成功后转染猪肺泡巨噬细胞3D4/2,通过Real-time PCR及Western blot验证My D88基因的表达水平,以及对LPS刺激后炎症因子TNF-α基因表达水平的影响。结果表明,所构建的猪My D88基因的特异性shRNA表达载体均可显著降低猪My D88 mRNA和蛋白质的表达水平(P0.05),干扰效率分别达到36%、67%、60%、69%;相比于未干扰组,LPS刺激My D88沉默之后的巨噬细胞,炎症因子TNF-α基因表达水平显著下降(P0.05),表明所构建猪My D88干扰载体干扰效果较好。     

奥利亚罗非鱼髓样分化因子(MyD88)的克隆及其在组织中的表达

髓样分化因子（myeloid differentiation factor 88,MyD88）是TLR（toll-like receptor）信号通路的关键接头蛋白,在先天性免疫中具有重要作用。通过RACE-RCR技术克隆了奥利亚罗非鱼（Oreochromis aureus）MyD88基因cDNA全长序列（GenBank登录号：JN032017）。序列分析表明,奥利亚罗非鱼MyD88 基因全长为1 611 bp,其中包括155 bp的5’非编码区,589 bp的3’非编码区和867 bp的编码区,编码288个氨基酸残基。MyD88蛋白N端具有死亡结构域,C端具有TIR结构域。同源性分析表明,奥利亚罗非鱼MyD88氨基酸序列与鳜鱼（Siniperca chuats）相似性最高,为85.8%,与其他鱼类相似性为70%~82%,与哺乳动物相似性为63%~66%;系统进化树分析表明,奥利亚罗非鱼MyD88与同属鲈形目的鳜鱼、大黄鱼（Larimichthys crocea）聚在一起。采用实时定量PCR方法检测MyD88在奥利亚罗非鱼各组织中的表达情况。结果显示,MyD88在所有被测组织中都有表达,其中表达量最高的是卵巢,其次在小肠、脾、肝、肾、鳃和血液中有较高的表达量,肌肉、精巢组织中表达量最低。本研究可为进一步探讨MyD88在奥利亚罗非鱼TLR信号通路中的作用奠定一定的基础。     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

Impaired Abdominal Wall Development and Deficient Wound Healing in Mice Lacking Aortic Carboxypeptidase-Like Protein

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.     

Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 mug/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-(14)C and uridine-5-(3)H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine.     

Glucose and Gluconate Metabolism in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.     

Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor β, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd^-/-), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in subpopulations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd^-/- mice. Furthermore, Pdgfd^-/- mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis.     

High Bone Mass in Mice Lacking Cx37 Because of Defective Osteoclast Differentiation

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37^−/−) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37^−/− mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37^−/− mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37^+/+ littermates. sRANKL/M-CSF treatment of nonadherent Cx37^−/− bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37^+/+ cell cultures. Further, Cx37^−/− osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37^−/− osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37^−/− mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.     

产肠毒素性大肠杆菌K88菌毛装配

K88菌毛介导产肠毒索性大肠杆菌在小肠上皮细胞的粘附,是引起新生仔猪腹泻的主要致病因子之一.菌毛的合成与装配是由fae操纵子调控的,fae操纵子包含10个基,faeA-fae J,其中有些基因表达菌毛装配所需的各种结构蛋白、分子伴侣和调控因子.菌毛的装配过程是由fae操纵子调控,通过分子伴侣,锚定蛋白的相互协同作用完成,组装成结构蛋白的多聚体.继阐明K88菌毛装配调控机理之后,K88菌毛在非毒素源性大肠杆菌及其它原核生物中装配也取得成功,同时菌毛结构蛋白在真核生物中组装也取得了很大进展.     

Defective Differentiation of Adipose Precursor Cells from Lipodystrophic Mice Lacking Perilipin 1

Perilipin 1 (Plin1) localizes at the surface of lipid droplets to regulate triglyceride storage and hydrolysis in adipocytes. Plin1 defect leads to low adiposity in mice and partial lipodystrophy in human. This study investigated the roles of Plin1 in adipocyte differentiation. Plin1 null (-/-) mice showed plenty of multilocular adipocytes and small unilocular adipocytes in adipose tissue, along with lack of a subpopulation of adipose progenitor cells capable of in vivo adipogenesis and along with downregulation of adipogenic pathway. Before initiation of differentiation, adipose stromal-vascular cells (SVCs) from Plin1-/- mice already accumulated numerous tiny lipid droplets, which increased in number and size during the first 12-h induction but thereafter became disappeared at day 1 of differentiation. The adipogenic signaling was dysregulated despite protein level of PPARγ was near normal in Plin1-/- SVCs like in Plin1-/- adipose tissue. Heterozygous Plin1+/- SVCs were able to develop lipid droplets, with both the number and size more than in Plin1-/- SVCs but less than in Plin1+/+ SVCs, indicating that Plin1 haploinsufficiency accounts for attenuated adipogenesis. Aberrant lipid droplet growth and differentiation of Plin1-/- SVCs were rescued by adenoviral Plin1 expression and were ameliorated by enhanced or prolonged adipogenic stimulation. Our finding suggests that Plin1 plays an important role in adipocyte differentiation and provides an insight into the pathology of partial lipodystrophy in patients with Plin1 mutation.     

Prognostic Value of Myeloid Differentiation Primary Response 88 and Toll-Like Receptor 4 in Breast Cancer Patients

Purpose

Breast cancer remains a major cause of death in women worldwide, and tumor metastasis is the leading cause of death in breast cancer patients after conventional treatment. Chronic inflammation is often related to the occurrence and growth of various malignancies. This study evaluated the prognosis of breast cancer patients based on contributors to the innate immune response: myeloid differentiation primary response 88 (MyD88) and Toll-like receptor 4 (TLR4).

Methods

We analyzed data from 205 breast invasive ductal carcinoma (IDC) patients who were treated at the Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Fudan University Shanghai Cancer Center, from 2002 to 2006. Overall survival (OS) and disease-free survival (DFS) were compared.

Results

In total, 152 patients (74.15%) were disease-free without relapse or metastasis, whereas 53 (25.85%) patients developed recurrence or metastasis. A significant positive correlation was observed between MyD88 and TLR4 expression (p<0.001). Patients with high expression were more likely to experience death and recurrence/metastasis events (p<0.05). Patients with low MyD88 or TLR4 expression levels had better DFS and OS than patients with high expression levels (log-rank test: p<0.001). Patients with low MyD88 and TLR4 expression levels had better DFS and OS than patients with high expression levels of either (log-rank test: p<0.001). In a multivariate analysis, high MyD88 expression was an independent predictive factor for decreased DFS (adjusted HR, 3.324; 95% CI, 1.663–6.641; p = 0.001) and OS (adjusted HR, 4.500; 95% CI, 1.546–13.098; p = 0.006).

Conclusions

TLR4-MyD88 signaling pathway activation or MyD88 activation alone may be a risk factor for poor prognosis in breast cancer. Therefore, TLR4-MyD88 signaling pathway activation in tumor biology provides a novel potential target for breast cancer therapy.     

Temperature-Sensitive Conjugation-Defective F Factor in Escherichia coli

A mutant strain of Escherichia coli K-12 which is unable to transfer genetic material at 42 C has been isolated. Although transfer is inhibited at this temperature, the formation of specific pairs is unaffected. This strain and its derivatives also show an altered sensitivity to certain ribonucleic acid-containing male specific phages at 42 C. Both phenotypic changes are reversed when a derepressed fi(+) R factor is present in the same cell and a revertant which has regained both activities has been obtained. The nature of the temperature-sensitive defect and its effect on the conjugation process are discussed.