MicroRNAs (miRNAs) are endogenously expressed 20 -24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.The first miRNA 1 was identified in Caenorhabditis elegans as a gene important for timing of larval development (5). miRNAs have since been implicated in many processes in invertebrates, including cell proliferation and apoptosis (6, 7), fat metabolism (6), and neuronal patterning (8). As many miRNAs are conserved across species (9 -11), they are likely to be involved in developmental processes in all animals. Only a few mammalian miRNAs have been assigned any function, and at least two of these are involved in developmental processes: miR-181 promotes B cell development in mice (12) and miR196a regulates several Hox genes (13), which code for a family of transcription factors involved in various developmental programs in animals (14).We hypothesized that miRNAs may play a role in maturation of human adipocytes. Understanding the molecular events involved in adipocyte differentiation is of interest for development of therapeutics for metabolic diseases such as obesity and diabetes. In vitro cell culture systems, such as human primary subcutaneous pre-adipocytes, have been crucial in uncovering signaling pathways important for adipocyte differentiation (15). These cells can be cultured with differentiation-promoting hormonal stimuli, causing them to develop into cells that morphologically and functionally resemble mature adipocytes. In this study we have inhibited a panel of miRNAs in pre-adipocytes using antisense oligonucleotides and evaluated the effect on adipocyte differentiation. Combined with expression analysis of miRNAs in differentiating adipocytes by microarray, one miRNA, miR-143, was identified which normally promotes adipocyte differentiation. These results indicate that miRNAs do play a role in adipocyte differentiation and are potential therapeutic targets for obesity and metabolic diseases. EXPERIMENTAL PROCEDURESOligonucleotide Synthesis-Oligonucleotides were prepared using conventional phosphoramidite chemistry and...

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH 2 -terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1 ؊/؊ cells. Consistently, Gab1؊/؊ cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.

The effect of a panel of proline mutants of dendroaspin, an inhibitor of platelet aggregation and cell adhesion, including A42-dendroaspin, A47-dendroaspin, A49-dendroaspin, A42,47-dendroaspin and A47,49-dendroaspin, was investigated using platelet-aggregation and cell-adhesion assays. Here we show that a single alanine-for-proline substitution did not affect potency when measured as the ability either to inhibit platelet aggregation induced by ADP (IC50 ≈ 170nM) or to block transfected A375-SM cell adhesion to fibrinogen in the presence of Mn2+ as compared with wild-type dendroaspin. By comparison, double proline substitution with alanines significantly reduced the potency in both assays by approx. 5–8-fold. These observations, therefore, suggest that proline residues flanking the RGD motif in dendroaspin and other RGD-containing venom proteins, e.g. disintegrins, may contribute to maintaining a favourable conformation for the solvent-exposed RGD site for its recognition by integrin receptors.

Anemia is one of the more common blood disorders and is associated with a number of diseases, including chronic kidney disease, chronic inflammation, and certain types of cancer. Under these conditions iron is essential as it is required for erythroid progenitor cell proliferation and red cell function. Hepcidin is a liver-derived growth factor that regulates iron absorption in the GI tract and iron absorption and release in tissues. Furthermore, hepcidin overexpression has been strongly linked mechanistically as a mediator of decreased iron availability and anemia. We have utilized an antisense approach to investigate the role of hepcidin in animal models of anemia and as a potential therapeutic approach for the treatment of this disorder in humans. Second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotides (ASOs) were screened in isolated primary mouse hepatocytes, followed by in vivo screening in mice, for the ability to reduce hepcidin mRNA levels. ASO treatment resulted in a reduction of hepcidin mRNA in liver which was associated with a significant increase in serum iron levels. The best hepcidin ASO was then tested in a mouse model of turpentine induced hypoferremia and anemia to determine the role of hepcidin in regulating serum iron and anemia endpoints. Mice were treated (I.P. twice/weekly) with hepcidin or control ASO for two weeks at varying doses prior to a single subcutaneous injection of turpentine. Turpentine treatment 16 hours post-injection produced a significant reduction in serum iron levels and at two weeks resulted in reduced RBC numbers, hematocrit and hemoglobin levels. Treatment with the hepcidin ASO resulted in a dose dependent improvement in all of these endpoints while the control oligonucleotide had no effect. Studies are in progress to further characterize the pharmacological activity of hepcidin ASO in additional models of anemia and results from these on-going studies will also be presented.