Cytoplasmic Drosha activity generated by alternative splicing

RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha.     

Dendritic cell immunogenicity is regulated by peroxisome proliferator-activated receptor gamma

Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-gamma during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-gamma activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-gamma activators. Moreover, activation of PPAR-gamma in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.     

T2384, a novel antidiabetic agent with unique peroxisome proliferator-activated receptor gamma binding properties

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma by TZDs improves insulin sensitivity; however, this is accompanied by the induction of several undesirable side effects. We have identified a novel synthetic PPARgamma ligand, T2384, to explore the biological activities associated with occupying different regions of the receptor ligand-binding pocket. X-ray crystallography studies revealed that T2384 can adopt two distinct binding modes, which we have termed "U" and "S", interacting with the ligand-binding pocket of PPARgamma primarily via hydrophobic contacts that are distinct from full agonists. The different binding modes occupied by T2384 induced distinct patterns of coregulatory protein interaction with PPARgamma in vitro and displayed unique receptor function in cell-based activity assays. We speculate that these unique biochemical and cellular activities may be responsible for the novel in vivo profile observed in animals treated systemically with T2384. When administered to diabetic KKAy mice, T2384 rapidly improved insulin sensitivity in the absence of weight gain, hemodilution, and anemia characteristics of treatment with rosiglitazone (a TZD). Moreover, upon coadministration with rosiglitazone, T2384 was able to antagonize the side effects induced by rosiglitazone treatment alone while retaining robust effects on glucose disposal. These results are consistent with the hypothesis that interactions between ligands and specific regions of the receptor ligand-binding pocket might selectively trigger a subset of receptor-mediated biological responses leading to the improvement of insulin sensitivity, without eliciting less desirable responses associated with full activation of the receptor. We suggest that T2384 may represent a prototype for a novel class of PPARgamma ligand and, furthermore, that molecules sharing some of these properties would be useful for treatment of type 2 diabetes.     

Dominant negative isoform of rat norepinephrine transporter produced by alternative RNA splicing

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3'-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a gamma-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.     

Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.     

A dominant negative peroxisome proliferator-activated receptor-gamma knock-in mouse exhibits features of the metabolic syndrome

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARgamma mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARgamma L466A mutation, shown previously to inhibit wild-type PPARgamma action in vitro. Homozygous PPARgamma L466A knock-in mice die in utero. Heterozygous PPARgamma L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARgamma function and the metabolic syndrome.