Ola Larsson

Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages

Attainment of a brown adipocyte cell phenotype in white adipocytes, with their abundant mitochondria and increased energy expenditure potential, is a legitimate strategy for combating obesity. The unique transcriptional regulators of the primary brown adipocyte phenotype are unknown, limiting our ability to promote brown adipogenesis over white. In the present work, we used microarray analysis strategies to study primary preadipocytes, and we made the striking discovery that brown preadipocytes demonstrate a myogenic transcriptional signature, whereas both brown and white primary preadipocytes demonstrate signatures distinct from those found in immortalized adipogenic models. We found a plausible SIRT1-related transcriptional signature during brown adipocyte differentiation that may contribute to silencing the myogenic signature. In contrast to brown preadipocytes or skeletal muscle cells, white preadipocytes express Tcf21, a transcription factor that has been shown to suppress myogenesis and nuclear receptor activity. In addition, we identified a number of developmental genes that are differentially expressed between brown and white preadipocytes and that have recently been implicated in human obesity. The interlinkage between the myocyte and the brown preadipocyte confirms the distinct origin for brown versus white adipose tissue and also represents a plausible explanation as to why brown adipocytes ultimately specialize in lipid catabolism rather than storage, much like oxidative skeletal muscle tissue.

mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs

Proliferation Control The protein complex mTORC1, which contains the protein kinase known as mammalian target of rapamycin, is an important regulator of cell proliferation and cell size. Among many targets, mTORC1 phosphorylates the eukaryotic translation initiation factor eIF4E–binding proteins (4E-BPs), thus controlling translation of proteins that regulate proliferation. Dowling et al. (p. 1172) used mice lacking expression of the 4E-BPs to show that these proteins contribute to mTORC1s activation of cell proliferation, but are dispensable for the effects of mTORC1 on cell growth. The latter required another mTORC1 target—the ribosomal protein S6 kinase. mTORC1 inhibitors are being explored as potential anticancer agents, and the presence of 4E-BPs was necessary for mTORC1 inhibitors to reduce the number and size of colonies formed by transformed mouse cells. Control of cell proliferation and cell size is separately regulated in mammals. The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogen and nutrient signals to control cell proliferation and cell size. Hence, mTORC1 is implicated in a large number of human diseases—including diabetes, obesity, heart disease, and cancer—that are characterized by aberrant cell growth and proliferation. Although eukaryotic translation initiation factor 4E–binding proteins (4E-BPs) are critical mediators of mTORC1 function, their precise contribution to mTORC1 signaling and the mechanisms by which they mediate mTORC1 function have remained unclear. We inhibited the mTORC1 pathway in cells lacking 4E-BPs and analyzed the effects on cell size, cell proliferation, and cell cycle progression. Although the 4E-BPs had no effect on cell size, they inhibited cell proliferation by selectively inhibiting the translation of messenger RNAs that encode proliferation-promoting proteins and proteins involved in cell cycle progression. Thus, control of cell size and cell cycle progression appear to be independent in mammalian cells, whereas in lower eukaryotes, 4E-BPs influence both cell growth and proliferation.

eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5′ cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development and progression. The activity of eIF4E is dysregulated in cancer. Regulation of eIF4E is partly achieved through phosphorylation. However, the physiological significance of eIF4E phosphorylation in mammals is not clear. Here, we show that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide analysis of translated mRNAs, we show that the phosphorylation of eIF4E is required for translational up-regulation of several proteins implicated in tumorigenesis. Accordingly, increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. Our findings establish eIF4E phosphorylation as a critical event in tumorigenesis. These findings raise the possibility that chemical compounds that prevent the phosphorylation of eIF4E could act as anticancer drugs.

mTORC1 Controls Mitochondrial Activity and Biogenesis through 4E-BP-Dependent Translational Regulation

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

Bactericidal antisense effects of peptide-PNA conjugates

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide–PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 μM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide–PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Translational control of the innate immune response through IRF-7

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-α and IFN-β), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Hypoxia-Independent Angiogenesis in Adipose Tissues during Cold Acclimation

The molecular mechanisms of angiogenesis in relation to adipose tissue metabolism remain poorly understood. Here, we show that exposure of mice to cold led to activation of angiogenesis in both white and brown adipose tissues. In the inguinal depot, cold exposure resulted in elevated expression levels of brown-fat-associated proteins, including uncoupling protein-1 (UCP1) and PGC-1alpha. Proangiogenic factors such as VEGF were upregulated, and endogenous angiogenesis inhibitors, including thrombospondin, were downregulated. In wild-type mice, the adipose tissues became hypoxic during cold exposure; in UCP1(-/-) mice, hypoxia did not occur, but, remarkably, the augmented angiogenesis was unaltered and was thus hypoxia independent. Intriguingly, VEGFR2 blockage abolished the cold-induced angiogenesis and significantly impaired nonshivering thermogenesis capacity. Unexpectedly, VEGFR1 blockage resulted in the opposite effects: increased adipose vascularity and nonshivering thermogenesis capacity. Our findings have conceptual implications concerning application of angiogenesis modulators for treatment of obesity and metabolic disorders.

Epigenetic Activation of a Subset of mRNAs by eIF4E Explains Its Effects on Cell Proliferation

Background Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5′ cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. Methodology/Principal Findings Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. Conclusions/Significance Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs.

Fibrotic extracellular matrix activates a profibrotic positive feedback loop

Pathological remodeling of the extracellular matrix (ECM) by fibroblasts leads to organ failure. Development of idiopathic pulmonary fibrosis (IPF) is characterized by a progressive fibrotic scarring in the lung that ultimately leads to asphyxiation; however, the cascade of events that promote IPF are not well defined. Here, we examined how the interplay between the ECM and fibroblasts affects both the transcriptome and translatome by culturing primary fibroblasts generated from IPF patient lung tissue or nonfibrotic lung tissue on decellularized lung ECM from either IPF or control patients. Surprisingly, the origin of the ECM had a greater impact on gene expression than did cell origin, and differences in translational control were more prominent than alterations in transcriptional regulation. Strikingly, genes that were translationally activated by IPF-derived ECM were enriched for those encoding ECM proteins detected in IPF tissue. We determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPF-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR-29. Our data support a model in which fibroblasts are activated to pathologically remodel the ECM in IPF via a positive feedback loop between fibroblasts and aberrant ECM. Interrupting this loop may be a strategy for IPF treatment.

Insulin Receptor Expression by Human Prostate Cancers

Although recent laboratory and population studies suggest that prostate cancer may be responsive to insulin, there is a gap in knowledge concerning the expression of insulin receptors on benign or malignant prostate tissue.