A genome-wide RNAi screen for modifiers of the circadian clock in human cells.

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

Clocks not winding down: unravelling circadian networks.

An intrinsic clock enables an organism to anticipate environmental changes and use energy sources more efficiently, thereby conferring an adaptive advantage. Having an intrinsic clock to orchestrate rhythms is also important for human health. The use of systems biology approaches has advanced our understanding of mechanistic features of circadian oscillators over the past decade. The field is now in a position to develop a multiscale view of circadian systems, from the molecular level to the intact organism, and to apply this information for the development of new therapeutic strategies or for enhancing agricultural productivity in crops.

High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase.

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

Emergence of noise-induced oscillations in the central circadian pacemaker.

Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.

Deletion of Gab1 in the liver leads to enhanced glucose tolerance and improved hepatic insulin action.

Insulin receptor substrate-1 (IRS-1) and IRS-2 are known to transduce and amplify signals emanating from the insulin receptor. Here we show that Grb2-associated binder 1 (Gab1), despite its structural similarity to IRS proteins, is a negative modulator of hepatic insulin action. Liver-specific Gab1 knockout (LGKO) mice showed enhanced hepatic insulin sensitivity with reduced glycemia and improved glucose tolerance. In LGKO liver, basal and insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 was elevated, accompanied by enhanced Akt/PKB activation. Conversely, Erk activation by insulin was suppressed in LGKO liver, leading to defective IRS-1 Ser612 phosphorylation. Thus, Gab1 acts to attenuate, through promotion of the Erk pathway, insulin-elicited signals flowing through IRS and Akt proteins, which represents a novel balancing mechanism for control of insulin signal strength in the liver.

Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.

The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBalpha and beta are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbalpha. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBalpha and beta function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.

Shp2 acts downstream of SDF-1alpha/CXCR4 in guiding granule cell migration during cerebellar development.

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1alpha or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1alpha in an in vitro cell migration assay, and SDF-1alpha treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1alpha/CXCR4 signal in guiding cerebellar granule cell migration.

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality.

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase.

Obesity and diabetes, termed "diabesity," are serious health problems that are increasing in frequency. However, the molecular mechanisms and neuronal regulation of these metabolic disorders are not fully understood. We show here that Shp2, a widely expressed Src homology 2-containing Tyr phosphatase, plays a critical role in the adult brain to control food intake, energy balance, and metabolism. Mice with a neuron-specific, conditional Shp2 deletion were generated by crossing a pan-neuronal Cre-line (CRE3) with Shp2(flox/flox) mice. These congenic mice, CRE3/Shp2-KO, developed obesity and diabetes and the associated pathophysiological complications that resemble those encountered in humans, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, diabetic nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis. This mouse model may help to elucidate the molecular mechanisms that lead to the development of diabesity in humans and provide a tool to study the in vivo complications of uncontrolled diabetes.

Concerted functions of Gab1 and Shp2 in liver regeneration and hepatoprotection.

Liver regeneration is a rapid and concerted response to injury, in which growth factor-generated intracellular signals result in activation of transcription factors, DNA synthesis, and hepatocyte proliferation. However, the link between cytoplasmic signals resulting in proliferative response to liver injury remains to be elucidated. We show here that association of Gab1 adaptor protein and Shp2 tyrosine phosphatase is a critical event at the early phase of liver regeneration. Partial hepatectomy (PH) rapidly and transiently induced assembly of a complex comprising Shp2 and tyrosine-phosphorylated Gab1 in wild-type hepatocytes. Consistently, liver-specific Shp2 knockout (LSKO) and liver-specific Gab1 knockout (LGKO) mice displayed very similar phenotypes of defective liver regeneration triggered by PH, including blunted extracellular signal-regulated kinase 1/2 (Erk1/2) activation, decreased expression of immediate-early genes, and reduced levels of cyclins A, E, and B1, as well as suppression of hepatocyte proliferation. In contrast, the Akt and interleukin-6/Stat3 pathways were up-regulated posthepatectomy in LSKO and LGKO mice, accompanied by improved hepatoprotection. Collectively, this study establishes the physiological significance of the Gab1/Shp2 link in promoting mitogenic signaling through the Erk pathway in mammalian liver regeneration.

Antitumor efficiency of the cytosine deaminase/5-fluorocytosine suicide gene therapy system on malignant gliomas: an in vivo study.

BACKGROUND: Suicide gene therapy, particularly that utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, represents a novel and attractive methodology of cancer research. Mechanistically, the CD enzyme can convert the antifungal agent 5-FC into the antimetabolite agent 5-fluorouracil (5-FU), thereby killing tumor cells. The purpose of this study was to investigate the antitumor efficiency of the CD/5-FC system in malignant gliomas using a nude mouse model. MATERIAL/METHODS: The eukaryotic expression plasmid pCMV-CD was transfected into U251 malignant glioma cells. Resistant clones (labeled U251/CD cells) were subsequently isolated and further confirmed by reverse transcription polymerase chain reaction (RT-PCR), immunofluoroscence, and immunoblot. Then U251/CD cells were incubated with 5-FC at various concentrations to measure viability ratios as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 5-FU concentrations in the media were measured by high-performance liquid chromatography (HPLC). Finally, the volumes and weights of tumors from glioma-bearing nude mice after 5-FC intervention were evaluated. RESULTS: The results revealed that the untreated U251 cells were insensitive to 5-FC whereas the U251/CD cells were highly sensitive. Apoptosis and cell death were observed on the U251/CD cells after 5-FC administration. HPLC analysis showed that 5-FU was detected in the U251/CD cell media. These in vivo animal data showed that the volumes and weights of the implanted tumors were dramatically decreased due to cell apoptosis and tumor necrosis. CONCLUSIONS: The in vivo results encourage a further investigation in a controlled trial on the treatment of malignant gliomas via the CD/5-FC gene therapy system.

Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis.

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)α. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction.

Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.

Intercellular coupling confers robustness against mutations in the SCN circadian clock network.

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals.

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.

Conditional deletion of Shp2 in the mammary gland leads to impaired lobulo-alveolar outgrowth and attenuated Stat5 activation.

Stat5 and Stat3, two members of the Stat (signal transducer and activator of transcription) family, are known to play critical roles in mammopoiesis/lactogenesis and involution, respectively, in the mammary gland. Phosphotyrosine phosphatase Shp2 has been shown to dephosphorylate and thus inactivate both Stat5 and Stat3 in vitro. Paradoxically, cell culture studies also suggest a positive role of Shp2 in promoting prolactin-stimulated Stat5 activation. We have shown here that selective deletion of Shp2 in mouse mammary glands suppresses Stat5 activity during pregnancy and lactation, resulting in significant impairment of lobulo-alveolar outgrowth and lactation. In contrast, Stat3 activity was slightly up-regulated shortly before/at involution, leading to normal epithelial cell apoptosis/involution in Shp2-deficient mammary gland. Thus, Shp2 acts to promote Stat5 activation by the JAK2.prolactin receptor complex, while negatively modulating Stat3 activity before the onset of involution. This is the first demonstration that Shp2 manipulates Stat5 and Stat3 activities reciprocally in mammary epithelial cells, providing novel insight into the complex mechanisms for regulation of various Stat family members by a cytoplasmic tyrosine phosphatase.

Shp2 is dispensable in the formation and maintenance of the neuromuscular junction.

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2(floxed/floxed) mice were crossed with mice expressing the Cre gene under the control of the human skeletal alpha-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR alpha subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.

Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism.

Shp2, a Src homology 2-containing tyrosine phosphatase, has been implicated in a variety of growth factor or cytokine signaling pathways. However, it is conceivable that this enzyme acts predominantly in one pathway versus the others in a cell, depending on the cellular context. To determine the putative functions of Shp2 in the adult brain, we selectively deleted Shp2 in postmitotic forebrain neurons by crossing CaMKIIalpha-Cre transgenic mice with a conditional Shp2 mutant (Shp2(flox)) strain. Surprisingly, a prominent phenotype of the mutant (CaMKIIalpha-Cre:Shp2(flox/flox) or CaSKO) mice was the development of early-onset obesity, with increased serum levels of leptin, insulin, glucose, and triglycerides. The mutant mice were not hyperphagic but developed enlarged and steatotic liver. Consistent with previous in vitro data, we found that Shp2 down-regulates Jak2/Stat3 (signal transducer and activator of transcription 3) activation by leptin in the hypothalamus. However, Jak2/Stat3 down-regulation is offset by a dominant Shp2 promotion of the leptin-stimulated Erk pathway, leading to induction rather than suppression of leptin resistance upon Shp2 deletion in the brain. Collectively, these results suggest that a primary function of Shp2 in postmitotic forebrain neurons is to control energy balance and metabolism, and that this phosphatase is a critical signaling component of leptin receptor ObRb in the hypothalamus. Shp2 shows potential as a neuronal target for pharmaceutical sensitization of obese patients to leptin action.

Identification of Shp-2 as a Stat5A phosphatase.

Stat5A, a member of the signal transducers and activators of transcription (Stat) family, is activated upon a single tyrosine phosphorylation. Although much is known about the activation process, the mechanism by which the tyrosine-phosphorylated Stat5A proteins are inactivated is largely unknown. In this report, we demonstrate that down-regulation of the tyrosine-phosphorylated Stat5A was via dephosphorylation. Using tyrosine-phosphorylated peptides derived from Stat5A, we were able to purify protein-tyrosine phosphatase Shp-2 from cell lysates. Shp-2, but not Shp-1, specifically interacted with Stat5A in vivo, and the interaction was tyrosine phosphorylation-dependent. Moreover, Shp-2 was able to accelerate Stat5A dephosphorylation, and dephosphorylation of Stat5A was dramatically delayed in Shp-2-deficient cells. Therefore, we conclude that Shp-2 is a Stat5A phosphatase, which down-regulates the active Stat5A in vivo.