Protein tyrosine phosphatase 1B (PTP1B) inhibits hepatic insulin signaling by dephosphorylating tyrosine residues in insulin receptor (IR) and insulin receptor substrate (IRS). MicroRNAs may modulate metabolic functions. In view of the lack of understanding of the regulatory mechanism of PTP1B and its chemical inhibitors, this study investigated whether dysregulation of specific microRNA causes PTP1B-mediated hepatic insulin resistance, and if so, what the underlying basis is. In high-fat-diet-fed mice or hepatocyte models with insulin resistance, the expression of microRNA-122 (miR-122), the most abundant microRNA in the liver, was substantially down-regulated among those predicted to interact with the 3 0 -untranslated region of PTP1B messenger RNA (mRNA). Experiments using miR-122 mimic and its inhibitor indicated that miR-122 repression caused PTP1B induction. Overexpression of cJun N-terminal kinase 1 (JNK1) resulted in miR-122 down-regulation with the induction of PTP1B. A dominant-negative mutant of JNK1 had the opposite effect. JNK1 facilitated inactivating phosphorylation of hepatocyte nuclear factor 4a (HNF4a) responsible for miR-122 expression, as verified by the lack of HNF4a binding to the gene promoter. The regulatory role of JNK1 in PTP1B induction by a decrease in miR-122 level was strengthened by cellbased assays using isoliquiritigenin and liquiritigenin (components in Glycyrrhizae radix) as functional JNK inhibitors; JNK inhibition enabled cells to restore IR and IRS1/2 tyrosine phosphorylation and insulin signaling against tumor necrosis factor alpha, and prevented PTP1B induction. Moreover, treatment with each of the agents increased miR-122 levels and abrogated hepatic insulin resistance in mice fed a high-fat diet, causing a glucose-lowering effect. Conclusion: Decreased levels of miR-122 as a consequence of HNF4a phosphorylation by JNK1 lead to hepatic insulin resistance through PTP1B induction, which may be overcome by chemical inhibition of JNK. (HEPATOLOGY 2012;56:2209-2220

The motor neuron (MN)-hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN-hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN-hexamer-bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN-hexamer, enhancing the transcriptional activity of the MN-hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN-hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.he combinatorial action of transcription factors is a prevalent strategy for achieving cellular complexity in the CNS. However, how the combinatorial action of transcription factors leads to the expression of distinct batteries of terminal differentiation genes, which together establish a specific cellular identity; how the cell fate-specifying transcription factors interact with extracellular cues remain unclear. To address these questions, it is essential to identify both the cis-regulatory elements in the genome, which recruit a specific combination of transcription factors, and the target genes associated with those cis-regulatory elements.One of the best examples of combinatorial transcription codes has emerged from studies of spinal motor neuron (MN) development (1). Two LIM-homeodomain (LIM-HD) factors, LIM homeobox 3 (Lhx3) and Islet-1 (Isl1), are vital for directing MN fate specification in the developing spinal cord (2-5). During this process, two Isl1:Lhx3 dimers bind to nuclear LIM interactor (NLI, also known as LDB for LIM domain binding) that has a self-dimerization domain, thereby forming the MN-hexamer complex ( Fig. 1A and Fig. S1A) (2, 6). The combinatorial expression of Lhx3 and Isl1 is capable of triggering MN specification in chick spinal cord, ES cells (ESCs), and induced pluripotent stem cells (2,(6)(7)(8). In contrast to MNs, during the specification of V2 interneurons, two Lhx3s and two NLIs form a tetrameric complex, which directs the V2-interneuron fate (Fig. S1A) (2, 9). Thus, the combinatorial action of Lhx3 and Isl1, via the formation of the MN-hexamer, is critical to determine MN identity over V2-interneuron fate. However, key questions remain unanswered. First, does the MN-hexamer directly control terminal differentiation genes that are required for consolidating the functional identity of MNs? Second, does the MN-hexamer collaborate with other transcription factors and/or extracellu...

During development, two cell types born from closely related progenitor pools often express identical transcriptional regulators despite their completely distinct characteristics. This phenomenon implies the need for a mechanism that operates to segregate the identities of the two cell types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). We demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing the MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate the V2a and MN pathways. Our study uncovers a widely applicable gene regulatory principle for segregating related cell fates.

The diamine putrescine and the polyamines (PAs), spermidine (Spd) and spermine (Spm), are ubiquitously occurring polycations associated with several important cellular functions, especially antisenescence. Numerous studies have reported increased levels of PA in plant cells under conditions of abiotic and biotic stress such as drought, high salt concentrations, and pathogen attack. However, the physiological mechanism of elevated PA levels in response to abiotic and biotic stresses remains undetermined. Transgenic plants having overexpression of SAMDC complementary DNA and increased levels of putrescine (1.4-fold), Spd (2.3-fold), and Spm (1.8-fold) under unstressed conditions were compared to wild-type (WT) plants in the current study. The most abundant PA in transgenic plants was Spd. Under salt stress conditions, enhancement of endogenous PAs due to overexpression of the SAMDC gene and exogenous treatment with Spd considerably reduces the reactive oxygen species (ROS) accumulation in intra- and extracellular compartments. Conversely, as compared to the WT, PA oxidase transcription rapidly increases in the S16-S-4 transgenic strain subsequent to salt stress. Furthermore, transcription levels of ROS detoxifying enzymes are elevated in transgenic plants as compared to the WT. Our findings with OxyBlot analysis indicate that upregulated amounts of endogenous PAs in transgenic tobacco plants show antioxidative effects for protein homeostasis against stress-induced protein oxidation. These results imply that the increased PAs induce transcription of PA oxidases, which oxidize PAs, which in turn trigger signal antioxidative responses resulting to lower the ROS load. Furthermore, total proteins from leaves with exogenously supplemented Spd and Spm upregulate the chaperone activity. These effects of PAs for antioxidative properties and antiaggregation of proteins contribute towards maintaining the physiological cellular functions against abiotic stresses. It is suggested that these functions of PAs are beneficial for protein homeostasis during abiotic stresses. Taken together, these results indicate that PA molecules function as antisenescence regulators through inducing ROS detoxification, antioxidative properties, and molecular chaperone activity under stress conditions, thereby providing broad-spectrum tolerance against a variety of stresses.

Salt stress causes rapid accumulation of nonexpressor of pathogenesis-related genes 1 (NPR1) protein, known as the redox-sensitive transcription coactivator, which in turn elicits many adaptive responses. The NPR1 protein transiently accumulates in chloroplast stroma under salt stress, which attenuates stress-triggered down-regulation of photosynthetic capability. We observed that oligomeric NPR1 in chloroplasts and cytoplasm had chaperone activity, whereas monomeric NPR1 in the nucleus did not. Additionally, NPR1 overexpression resulted in reinforcement of morning-phased and evening-phased circadian clock. NPR1 overexpression also enhanced antioxidant activity and reduced stress-induced reactive oxygen species (ROS) generation at early stage, followed with transcription levels for ROS detoxification. These results suggest a functional switch from a molecular chaperone to a transcriptional coactivator, which is dependent on subcellular localization. Our findings imply that dual localization of NPR1 is related to proteostasis and redox homeostasis in chloroplasts for emergency restoration as well as transcriptional coactivator in the nucleus for adaptation to stress.Chloroplasts are particularly vulnerable to environmental disturbances, because of oxygenic photosynthesis 1 , after which the generation of reactive oxygen species (ROS) 2 might occur as a more serious phenomenon 3 . Even though ROS play an important role as signaling molecules and inducers in the adaption of plants to abiotic stress, they are also toxic byproducts of stress metabolism 4 . Chloroplasts act as sensors of the present environmental situation 5 and produce diverse signals communicating the functionality of the photosynthetic apparatus to the nucleus, which is defined as retrograde signaling 6 .Recent genomic technologies provide growing evidence that ROS generation is one of the most common responses to different stresses in plants, representing various signaling pathways come together 7,8 . Because the rapid generation of ROS represents a common plant response to almost all environmental challenges 4,9 , it is suggested that ROS and the redox system in chloroplasts represent primary sources within the plant signaling battery. This hypothesis implies there are interactions between ROS and other signaling components 4 such as redox homeostasis, plant hormones, and transcription factors 4 .Sunlight for photosynthesis is available only for a limited period within the 24 h day. The rhythmic and predictable alteration of solar energy has driven the evolution of the circadian clock, which is integrated with signals within chloroplasts 4,10 . Nuclear-encoded transcripts for chloroplast proteins may be related to the circadian regulation of chloroplasts 4,11 .Proper protein folding and localization are critical for cellular protein function. However, cells are exposed to environmental stresses, which makes them susceptive to nonnative condition that ultimately can result in misfolding and aggregation 4,12 . In addition, ROS or oxidized small mole...