Yaou Zhang

MiRNA-Directed Regulation of VEGF and Other Angiogenic Factors under Hypoxia

MicroRNAs (miRNAs) are a class of 20–24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle). We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false positive miRNAs might be useful strategies in the avoidance of unwanted cross-action among genes targeted by miRNAs with multiple targets.

Deficiency of cathepsin S reduces atherosclerosis in LDL receptor–deficient mice

Human atherosclerotic lesions overexpress the lysosomal cysteine protease cathepsin S (Cat S), one of the most potent mammalian elastases known. In contrast, atheromata have low levels of the endogenous Cat S inhibitor cystatin C compared with normal arteries, suggesting involvement of this protease in atherogenesis. The present study tested this hypothesis directly by crossing Cat S-deficient (CatS(-/-)) mice with LDL receptor-deficient (LDLR(-/-)) mice that develop atherosclerosis on a high-cholesterol diet. Compared with LDLR(-/-) mice, double-knockout mice (CatS(-/-)LDLR(-/-)) developed significantly less atherosclerosis, as indicated by plaque size (plaque area and intimal thickening) and stage of development. These mice also had markedly reduced content of intimal macrophages, lipids, smooth muscle cells, collagen, CD4(+) T lymphocytes, and levels of IFN-gamma. CatS(-/-)LDLR(-/-) monocytes showed impaired subendothelial basement membrane transmigration, and aortas from CatS(-/-)LDLR(-/-) mice had preserved elastic laminae. These findings establish a pivotal role for Cat S in atherogenesis.

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17∼92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

Macrophage Migration Inhibitory Factor Deficiency Impairs Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

Background— Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed widely by vascular cells. However, scant in vivo evidence supports direct participation of MIF in atherogenesis. Therefore, we investigated whether deficiency of MIF modulates atherosclerotic lesion formation and composition in low-density lipoprotein receptor–deficient (LDLr−/−) mice. Methods and Results— MIF−/−LDLr−/− and LDLr−/− mice were generated and consumed an atherogenic diet for 12 or 26 weeks. MIF−/−LDLr−/− mice had significantly reduced abdominal aorta lipid deposition and intimal thickening from aortic arch throughout the abdominal aorta compared with LDLr−/− mice. Marked retardation of atherosclerosis over time in MIF-deficient mice accompanied decreased lesion cell proliferation. At 26 weeks, 20% of MIF-deficient mice developed only early, fatty streak–like lesions, whereas >80% of LDLr−/− mice developed advanced lesions containing calcification and lipid cores. Analysis of smooth muscle cells from mouse aortae demonstrated that MIF deficiency reduced smooth muscle cell proliferation, cysteine protease expression, and elastinolytic and collagenolytic activities. Conclusions— Deficiency of MIF reduces atherogenesis in LDLr−/− mice. These results provide novel insight into inflammatory pathways operating in atheromata and identify a new potential target for modulating atherogenesis.

Cathepsin L activity controls adipogenesis and glucose tolerance.

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R β-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR β-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR β-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders.

The G3 Domain of Versican Enhances Cell Proliferation via Epidermial Growth Factor-like Motifs

Versican is a member of the large aggregating chondroitin sulfate proteoglycan family. We have expressed in NIH3T3 fibroblasts a recombinant versican mini-gene comprising the G1 and G3 domains and 15% of the CS domain. We observed that expression of the mini-versican gene stimulated cell proliferation as determined by cell counting and cell cycle analysis. Addition of exogenous mini-versican protein to cultured cells produced the same result. The effects of the mini-versican were greatly reduced when the G3 domain was deleted. Expression of the G3 domain alone promotes cell proliferation, and addition of purified G3 gene products to NIH3T3 fibroblasts and cultured chicken fibroblasts enhances cell growth. Further, deletion of the epidermal growth factor (EGF)-like motifs in the versican G3 domain reduced the effects of the mini-versican on cell proliferation. In the presence of the purified mini-versican protein, antisense oligonucleotides to the EGF receptor inhibited proliferation of NIH3T3 fibroblasts, compared with control sense oligonucleotides. Taken together, these results imply that versican enhances cell proliferation, and this effect is mediated, at least in part, by the action of versican EGF-like motifs on endogenous EGF receptor.

MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7

MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3′-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3′-untranslated region (3′UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3′UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3′UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation.

MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

Cystatin C Deficiency Increases Elastic Lamina Degradation and Aortic Dilatation in Apolipoprotein E-Null Mice

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. The lysosomal cysteine proteases can exert potent elastolytic and collagenolytic activity. Human atherosclerotic plaques have increased cysteine protease content and decreased levels of the endogenous inhibitor cystatin C, suggesting an imbalance that would favor matrix degradation in the arterial wall. This study tested directly the hypothesis that impaired expression of cystatin C alters arterial structure. Cystatin C–deficient mice (Cyst C−/−) were crossbred with apolipoprotein E–deficient mice (ApoE−/−) to generate cystatin C and apolipoprotein E–double deficient mice (Cyst C−/−ApoE−/−). After 12 weeks on an atherogenic diet, cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation, decreased medial size, and increased smooth muscle cell and collagen content in aortic lesions of ApoE−/− mice. Cyst C−/−ApoE−/− mice also showed dilated thoracic and abdominal aortae compared with control ApoE−/− mice, although atheroma lesion size, intimal macrophage accumulation, and lipid core size did not differ between these mice. These findings demonstrate directly the importance of cysteine protease/protease inhibitor balance in dysregulated arterial integrity and remodeling during experimental atherogenesis.

β1-Integrin–collagen interaction reduces chondrocyte apoptosis

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte–matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin β1 suggest that the collagen–chondrocyte interactions are mediated through integrin β1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte–matrix network.