Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

Epigallocatechin-3-gallate对原代大鼠脂肪细胞脂肪分解及HSL、TGH转录表达的影响

体内能量代谢是维持机体正常生理活动的基础,而脂肪细胞的脂解是能量代谢的核心反应之一,调控脂肪酸从TG库释放,后由血清白蛋白转运至体内各个组织以满足能量需要。如果脂解作用出现障碍,就会影响机体能量的平衡进而引发肥胖和胰岛素抵抗等疾病。表没食子儿茶素没食子酸酯(Epig     

Post-translational modifications of collagen upon BMP-induced osteoblast differentiation

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24 h of treatment, the expression of most isoforms were upregulated up to 96 h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

Regulation of ATGL expression mediated by leptin in vitro in porcine adipocyte lipolysis

Adipose triglyceride lipase (ATGL), as an adipose-enriched protein, is able to hydrolyze triglycerides and plays an important part in triglyceride lipolysis of fat tissue. Leptin, an adipocyte cytokine, can increase the fat decomposition process. Many phenomena indicate that ATGL has a close relationship with leptin’s promoting the hydrolysis of triglycerides. However, the regulatory mechanism of ATGL in leptin’s promoting fat hydrolysis has not been directly and systematically studied yet. This study demonstrated that ATGL was expressed in vitro by leptin regulation. The amount of ATGL mRNA increased and the amount of ATGL protein decreased based on a dose-dependent manner when leptin concentrations ranged from 5 to 50 ng/ml were used to treat fully differentiated porcine adipocytes for 3 h. In addition, this study revealed that JAK-STAT and MAPK signaling pathways, as well as PPARγ all played important roles in the ATGL expression mediated by leptin.     

The central role of perilipin a in lipid metabolism and adipocyte lipolysis

The related disorders of obesity and diabetes are increasing to epidemic proportions. The role of neutral lipid storage and hydrolysis, and hence the adipocyte, is central to understanding this phenomenon. The adipocyte holds the major source of stored energy in the body in the form of triacylglycerols (TAG). It has been known for over 35 years that the breakdown of TAG and release of free (unesterified) fatty acids and glycerol from fat tissue can be regulated by a cAMP-mediated process. However, beyond the initial signaling cascade, the mechanistic details of this lipolytic reaction have remained unclear. Work in recent years has revealed that both hormone-sensitive lipase (HSL), generally thought to be the rate-limiting enzyme, and perilipin, a lipid droplet surface protein, are required for optimal lipid storage and fatty acid release. There are multiple perilipin proteins encoded by mRNA splice variants of a single perilipin gene. The perilipin proteins are polyphosphorylated by protein kinase A and phosphorylation is necessary for translocation of HSL to the lipid droplet and enhanced lipolysis. Hence, the surface of the lipid storage droplet has emerged as a central site of regulation of lipolysis. This review will focus on adipocyte lipolysis with emphasis on hormone signal transduction, lipolytic enzymes, the lipid storage droplet, and fatty acid release from the adipocyte.     

串联亲和标签LOX蛋白慢病毒表达载体的构建及在乳腺癌细胞中的表达

目的:为了研究赖氨酰氧化酶(Lysyl Oxidase,LOX)及其相互作用蛋白质在乳腺癌中的功能,构建带StrepⅡ/FLAG串联亲和标签的重组LOX蛋白慢病毒表达载体并在乳腺癌细胞MDA-MB-231中表达。方法:设计引物通过聚合酶链式反应获得带StrepⅡ/FLAG串联亲和标签的LOX蛋白(LOX-SF)的亲本质粒,双酶切后鉴定测序克隆至GV303表达载体,连同慢病毒包装质粒共同转染293T得到GV303/LOX-SF慢病毒,将其转染MDA-MB-231细胞,使用荧光定量PCR和蛋白质印迹实验对细胞中重组蛋白LOX-SF进行检测。结果:通过串联亲和纯化获得LOX-SF重组蛋白,使用标签抗体成功鉴定到LOX-SF重组蛋白在MDA-MB-231细胞内稳定表达。结论:GV303/LOX-SF的构建,使带StrepⅡ/FLAG融合标签的LOX蛋白在MDA-MB-231中成功表达及纯化,为筛选和研究LOX及其相互作用蛋白在乳腺癌细胞内的功能奠定了实验基础。     

心脏特异表达LMNA^E82K转基因小鼠的建立

目的建立心脏特异表达LMNAE82K转基因小鼠,为研究LMNAE82K与心肌病发病机制的关系提供工具动物。方法把LMNAE82K基因插入α-MHC启动子下游,构建转基因表达载体,显微注射法建立C57BL/6JLMNAE82K转基因小鼠,PCR鉴定转基因小鼠的基因型,采用Western Blot鉴定LMNAE82K在心脏组织中的表达,H＆E染色和超声检测转基因小鼠心脏的病理改变。结果建立了2个心脏组织特异表达LMNAE82K的转基因小鼠品系。超声检查显示转基因小鼠心室壁变薄,收缩期容积和舒张期容积增加,射血分数及短轴缩短率降低。结论LMNAE82K转基因小鼠具有LMNAE82K引起的家族性扩心病有类似的病理变化,为研究LMNAE82K与心肌病发病机制的关系的研究提供了有价值的疾病动物模型。     

The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis

This study assessed the effects of selective inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 +/- 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 +/- 25% and 235 +/- 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 +/- 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 +/- 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 +/- 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.     

The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult

Lysyl hydroxylase catalyzes the hydroxylation of lysine residues in collagenous sequences. Three isoforms (LH1, LH2 and LH3) of lysyl hydroxylase have been characterized, and LH2 is present as two alternatively spliced forms. In order to better understand the functional differences between the isoforms in vivo, the expression of the different isoforms was studied in mouse embryos and adult tissues. Our data indicate a widespread expression of all isoforms during embryogenesis, whereas the expression profiles become more specialized in adult tissues. The expression of LH2 was more tissue-specific, whereas a uniform and housekeeping like behavior was observed for LH3. Some cells express both LH2 and LH3, while a clear cell specificity was seen in some tissues. Moreover, immunoelectron microscopy revealed differences in the localization of LH2 and LH3. LH2 was localized intracellularly in the ER in all tissues studied, whereas the localization of LH3 was either intracellular or extracellular or both, depending on the tissue. Furthermore, our data indicate that the alternative splicing of LH2 is developmentally regulated. The short form of LH2 (LH2a) is the predominant form until E11.5; the long form (LH2b) dominates thereafter and is the major form in many adult tissues. Interestingly, however, adult mouse kidney and testis express exclusively the short form, LH2a. The results reveal a specific regulation for the expression of LH isoforms as well as for alternative splicing of LH2 during embryogenesis and in different tissues.     

赖氨酰氧化酶样蛋白2在肾细胞癌中的表达及其临床意义

为探究赖氨酰氧化酶样蛋白2 (lysyl oxidase like 2, LOXL2)与肾细胞癌(renal cell carcinoma, RCC)的关系,本研究采用免疫组织化学方法检测58例RCC组织及13例癌旁组织中LOXL2的表达水平,并利用统计学方法进一步分析LOXL2与临床病理参数和预后之间的关系。研究显示,LOXL2在癌组织和癌旁组织的阳性表达率分别为68.96%和23.08%,差异具有统计学意义(p<0.05)。LOXL2阳性表达与患者的临床分期和肾包膜浸润相关(p<0.05),但是与患者的性别、年龄、吸烟、病理类型、肿瘤大小和肿瘤分级均无相关性(p>0.05)。采用Kaplan-Meier法和Log-Rank检验分析LOXL2与术后生存时间的关系,实验显示LOXL2阳性组和阴性组患者的5年生存率分别为61.58%和89.46%,差异具有统计学意义(p<0.05)。我们的研究表明LOXL2在肾癌中过表达,其可能在肾癌的侵袭和转移过程中发挥作用,可作为预后的分子标记物。     

An analytical comparison of cobalt cardiomyopathy and idiopathic dilated cardiomyopathy

Toxic cardiomyopathy (TC) has a rapid clinical course and morphologically resembles idiopathic dilated cardiomyopathy (IDC). To further characterize TC, we used light microscopy to compare lesions caused by cobalt (Co) to those of IDC. Cobalt levels were also measured as a chemical marker to differentiate TC from IDC. We reviewed cases with TC and IDC and excluded all cases with chemotherapy-induced myopathy and catecholamine toxicity as well as cases with possible infectious, ischemic, or hypersensitivity-induced myopathies. We compared the light microscopic findings of 12 TC cases to 12 cases of IDC, and measured trace Co levels on digested heart tissue samples. The TC cases had prominent myofibrillar loss and atrophy; no cases had neutrophil infiltration or frank myocyte necrosis. In contrast, IDC had minimal myofibril loss and atrophy. Cobalt levels in the range of 0.6 to 5.45 μg/g of dry tissue were obtained for the TC cases, while IDC demonstrated Co levels of 0.01–0.2 μg/g. Distinction between TC and IDC is predominantly a function of myocyte change, with TC showing myofibrillar loss and atrophy in the absence of inflammatory infiltrates and fibrosis; IDC is predominantly associated with myocyte hypertrophy, atrophy, and fibrosis. The opinions or assertions expressed herein are the private views of the authors and are not to be construed as official or as representing the views of the Armed Forces Institute of Pathology, the Department of the Army or the Department of Defense.     

Crosslinked elastic fibers are necessary for low energy loss in the ascending aorta

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln^−/−) or two key proteins (lysyl oxidase, Lox^−/−, or fibulin-4, Fbln4^−/−) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln^−/−, Lox^−/−, and Fbln4^−/− ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56–97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln^−/− aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53–387% in Eln^−/−, Lox^−/−, and Fbln4^−/− aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.     

The chemistry of natural enzyme-induced cross-links of proteins

Summary The cross-linking of protein molecules to form stable supramolecular aggregates capable of acting as protective and supporting structures is a common feature of organisms coping with the stresses of life. These new polymeric forms range from thick rigid structures to thin flexible membranes. The formation of such cross-links must be carefully controlled since more or less than optimal cross-linking could lead to malfunction or even death of the organism. The chemistry of the amino acids converted or directly involved in the formation of these cross-links is complex and a range of new amino acids has been identified. Di- and tri-tyrosines are formed by the action of peroxidases, quinones by catechol oxidases, glutamyl lysine iso-peptide bonds by glutamyl transferase and a complex series of lysine- aldehyde derived cross-links induced by lysyl oxidase. These cross-linking mechanisms provide an insight into the complex changes in tissue function during growth of the organism and their effects on the properties of foods.     

心脏特异表达Calponin 1转基因小鼠的心脏功能分析

目的建立心脏特异表达Calponin 1转基因小鼠,研究Calponin 1对心脏发育及心肌病的调节作用。方法利用心脏特异启动子α-MHC构建转基因表达载体,显微注射法建立Calponin 1转基因小鼠,PCR法鉴定转基因小鼠的基因型,Western Blot检测Calponin 1在心脏组织中的表达,心脏超声检测转基因小鼠的心脏结构和功能,HE染色和Masson染色检测转基因小鼠心脏的病理改变。结果 Calponin 1在野生型小鼠心脏中有表达,在扩张型心肌病小鼠的心脏组织表达降低。通过显微注射法,建立了2个心脏组织Calponin 1基因高表达的转基因小鼠系。与野生型小鼠相比,Calponin 1转基因小鼠收缩期左室内径（LVID,systolic）增加28%（P〈0.01,n=12）,舒张期左室内径（LVID,diastolic）增加16.2%（P〈0.01,n=12）,收缩期左室后壁厚度（LVPW,systolic）减小15.7%（P〈0.01,n=12）,舒张期左室后壁厚度（LVPW,diastolic）减小21%（P〈0.01,n=12）,射血分数（ejection fraction,EF）降低11.5%（P〈0.01,n=12）,短轴内径缩短率（fraction shortening,FS）降低14.6%（P〈0.05,n=12）。转基因小鼠心脏组织病理H＆E染色和Masson染色显示,转基因小鼠心室扩张,心肌细胞不均匀肥大,细胞间隙变大,心肌间质纤维增多。结论 Calponin 1在心脏特异过表达引起转基因小鼠心脏左室内径增加,收缩期容积和舒张期容积显著增大,心室壁变薄,射血分数及短轴缩短率降低等扩张性心肌病表型,推测Calponin 1是参与心肌病病理发生的基因之一。     

TNF-related apoptosis-inducing ligand and its decoy receptor osteoprotegerin in nonischemic dilated cardiomyopathy

Apoptosis has been attributed an essential role in dilated cardiomyopathy (DCM) recently. We assessed expression of TNF-related apoptosis-inducing ligand (TRAIL) and its decoy receptor osteoprotegerin (OPG) in men with nonischemic DCM, who underwent coronary angiography and endomyocardial biopsy (EMB) after exclusion of coronary artery disease compared to control patients. TRAIL plasma concentrations were elevated in DCM (p=0.02 vs. controls), and were positively correlated with left ventricular enddiastolic diameter (r=0.15, p=0.04), whereas OPG plasma levels did not differ between both groups (p=0.96). In EMB of DCM patients, TRAIL and OPG protein were detected by immunohistochemistry but not in controls. Furthermore, gene expression in EMB or peripheral blood leukocytes (PBL) of DCM patients assessed by real-time PCR showed an increase of TRAIL mRNA in PBL (p=0.01 vs. controls), whereas OPG mRNA was upregulated in endomyocardial specimens (p<0.001 vs. controls). In conclusion, myocardial overexpression of antiapoptotic OPG in DCM patients may represent a compensatory mechanism to limit systemic activation of TRAIL in patients with congestive heart disease.     

Essential role of lysyl oxidases in notochord development

Recent studies reveal a critical role for copper in the development of the zebrafish notochord, suggesting that specific cuproenzymes are required for the structural integrity of the notochord sheath. We now demonstrate that beta-aminopropionitrile, a known inhibitor of the copper-dependent lysyl oxidases, causes notochord distortion in the zebrafish embryo identical to that seen in copper deficiency. Characterization of the zebrafish lysyl oxidase genes reveals eight unique sequences, several of which are expressed in the developing notochord. Specific gene knockdown demonstrates that loss of loxl1 results in notochord distortion, and that loxl1 and loxl5b have overlapping roles in notochord formation. Interestingly, while notochord abnormalities are not observed following partial knockdown of loxl1 or loxl5b alone, in each case this markedly sensitizes developing embryos to notochord distortion if copper availability is diminished. Likewise, partial knockdown of the lysyl oxidase substrate col2a1 results in notochord distortion when combined with reduced copper availability or partial knockdown of loxl1 or loxl5b. These data reveal a complex interplay of gene expression and nutrient availability critical to notochord development. They also provide insight into specific genetic and nutritional factors that may play a role in the pathogenesis of structural birth defects of the axial skeleton.