Yanbin Zheng

Improved insulin sensitivity by calorie restriction is associated with reduction of ERK and p70S6K activities in the liver of obese Zucker rats

Calorie restriction (CR) improves obesity-related insulin resistance through undefined molecular mechanisms. Insulin receptor substrate (IRS)-1 serine/threonine kinases have been proposed to modulate insulin sensitivity through phosphorylation of IRS proteins. The aim of this study is to test the hypothesis that changes in the activity of IRS1 serine/threonine kinases may underlie the molecular mechanism of CR in improving insulin sensitivity. Obese and lean Zucker rats were subjected to 40% CR or allowed to feed ad libitum (AL) for 20 weeks; body weight and insulin sensitivity were monitored throughout this period. The activity of IRS1 serine/threonine kinases - including JNK, ERK, MTOR/p70(S6K) (RPS6KB1 as listed in the MGI Database), glycogen synthase kinase 3beta (GSK3B), AMPK (PRKAA1 as listed in the MGI Database), and protein kinase C (PRKCQ) in liver tissue extracts was measured by an in vitro kinase assay using various glutathione-S-transferase (GST)-IRS1 fragments as substrates, while phosphorylation of IRS1 and serine kinases was determined by western blotting using phosphospecific antibodies. CR in obese rats significantly reduced body weight and increased insulin sensitivity compared to AL controls. Serine kinase activity toward IRS1(S612) (corresponding to S616 in human IRS1) and IRS1(S632/635) (corresponding to S636/639 in human IRS1) was increased in obese rats compared to lean littermates, and was markedly decreased following CR. Concomitantly, obesity increased and CR decreased the activity of hepatic ERK and p70(S6K) against IRS1. The close association between the activity of hepatic ERK and p70(S6K) with insulin resistance suggests an important role for ERK and p70(S6K) in the development of insulin resistance, presumably via phosphorylation of IRS proteins.

Glycogen synthase kinase 3β mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulins intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3beta (GSK3 beta or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine(332) phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine(332) to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3 beta was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3 beta contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

Tgfβ signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK

We have investigated how the Arf gene product, p19Arf, is activated by Tgfβ during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfβ signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfβ depend on Arf in vivo. Tgfβ1, -2, and -3 (but not BMP-2, another member of the Tgfβ superfamily) induce p19Arf expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in ArflacZ/lacZ MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfβ type I receptor (TβrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfβ2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfβ2 effects, as does Cre recombinase treatment of Tgfbr2fl/fl MEFs to delete Tgfβ receptor II. Chromatin immunoprecipitation reveals that Tgfβ rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1β. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19Arf increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TβrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.

Dephosphorylation by Default, a Potential Mechanism for Regulation of Insulin Receptor Substrate-1/2, Akt, and ERK1/2

Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.

p19Arf represses platelet-derived growth factor receptor β by transcriptional and posttranscriptional mechanisms

ABSTRACT In addition to cancer surveillance, p19Arf plays an essential role in blocking signals stemming from platelet-derived growth factor receptor β (Pdgfrβ) during eye development, but the underlying mechanisms have not been clear. We now show that without Arf, pericyte hyperplasia in the eye results from enhanced Pdgfrβ-dependent proliferation from embryonic day 13.5 (E13.5) of mouse development. Loss of Arf in the eye increases Pdgfrβ expression. In cultured fibroblasts and pericyte-like cells, ectopic p19Arf represses and Arf knockdown enhances the expression of Pdgfrβ mRNA and protein. Ectopic Arf also represses primary Pdgfrβ transcripts and a plasmid driven by a minimal promoter, including one missing the CCAAT element required for high-level expression. p19Arf uses both p53-dependent and -independent mechanisms to control Pdgfrβ. In vivo, without p53, Pdgfrβ mRNA is elevated and eye development abnormalities resemble the Arf −/− phenotype. However, effects of p53 on Pdgfrβ mRNA do not appear to be due to direct p53 or RNA polymerase II recruitment to the promoter. Although p19Arf controls Pdgfrβ mRNA in a p53-dependent manner, it also blunts Pdgfrβ protein expression by blocking new protein synthesis in the absence of p53. Thus, our findings demonstrate a novel capacity for p19Arf to control Pdgfrβ expression by p53-dependent and -independent mechanisms involving RNA transcription and protein synthesis, respectively, to promote the vascular remodeling needed for normal vision.

Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.

Arf Induction by Tgfβ Is Influenced by Sp1 and C/ebpβ in Opposing Directions

Recent studies show that Arf, a bona fide tumor suppressor, also plays an essential role during mouse eye development. Tgfβ is required for Arf promoter activation in developing mouse eyes, and its capacity to induce Arf depends on Smads 2/3 as well as p38 Mapk. Substantial delay between activation of these pathways and increased Arf transcription imply that changes in the binding of additional transcription factors help orchestrate changes in Arf expression. Focusing on proteins with putative DNA binding elements near the mouse Arf transcription start, we now show that Tgfβ induction of this gene correlated with decreased expression and DNA binding of C/ebpβ to the proximal Arf promoter. Ectopic expression of C/ebpβ in mouse embryo fibroblasts (MEFs) blocked Arf induction by Tgfβ. Although basal levels of Arf mRNA were increased by C/ebpβ loss in MEFs and in the developing eye, Tgfβ was still able to increase Arf, indicating that derepression was not the sole factor. Chromatin immunoprecipitation (ChIP) assay showed increased Sp1 binding to the Arf promotor at 24 and 48 hours after Tgfβ treatment, at which time points Arf expression was significantly induced by Tgfβ. Chemical inhibition of Sp1 and its knockdown by RNA interference blocked Arf induction by Tgfβ in MEFs. In summary, our results indicate that C/ebpβ and Sp1 are negative and positive Arf regulators that are influenced by Tgfβ.

Negative regulation of initial steps in skeletal myogenesis by mTOR and other kinases

The transition from a committed progenitor cell to one that is actively differentiating represents a process that is fundamentally important in skeletal myogenesis. Although the expression and functional activation of myogenic regulatory transcription factors (MRFs) are well known to govern lineage commitment and differentiation, exactly how the first steps in differentiation are suppressed in a proliferating myoblast is much less clear. We used cultured mammalian myoblasts and an RNA interference library targeting 571 kinases to identify those that may repress muscle differentiation in proliferating myoblasts in the presence or absence of a sensitizing agent directed toward CDK4/6, a kinase previously established to impede muscle gene expression. We identified 55 kinases whose knockdown promoted myoblast differentiation, either independently or in conjunction with the sensitizer. A number of the hit kinases could be connected to known MRFs, directly or through one interaction node. Focusing on one hit, Mtor, we validated its role to impede differentiation in proliferating myoblasts and carried out mechanistic studies to show that it acts, in part, by a rapamycin-sensitive complex that involves Raptor. Our findings inform our understanding of kinases that can block the transition from lineage commitment to a differentiating state in myoblasts and offer a useful resource for others studying myogenic differentiation.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn , were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G 0 /G 1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.

Abstract 1097: Arf induction by Tgf-beta2 is mediated by Smad-2/3 dependent enhancement of Arf transcription

The Arf tumor suppressor gene product, p19Arf, is induced in response to certain oncogenic signals to block cancer development and progression. How the oncogenic cues are coupled to Arf promoter activation remains poorly defined, though. We took advantage of the fact that the Arf promoter is strictly controlled during development to investigate the normal regulatory mechanisms. We have recently published that Tgf-beta2 is required for Arf promoter activation at several sites in the developing mouse. To define mechanisms for the regulation, we developed a cell culture-based assay. We now show that Tgf-beta2 (5 ng/ml) induced p19Arf expression in WT mouse embryo fibroblasts (MEFs), enhanced Arf promoter activity in Arf lacZ/lacZ MEFs, and maintained a G1 cell cycle arrest in an Arf-dependent manner. The Arf promoter was also increased by Tgf-beta1, which functionally overlaps with Tgf-beta2, but not by Pdgf-B, a potent mitogen, nor BMP-2, another Tgf-beta superfamily member. To elucidate which pathway mediated the Tgf-beta2 effects, we used a panel of chemical inhibitors of Smad-dependent and -independent pathways. Among them, SB431542, a Tgf-beta type I receptor inhibitor, blocked Smad2 phosphorylation and impeded Tgf-beta2 induction of Arf. Genetic studies confirmed this because RNAi-driven knock-down of Tgf-beta receptor I, Smad2 or Smad3 impaired ArflacZ induction by Tgf-beta2. Further, Tgf-beta2 failed to induce p19Arf in Cre-treated Tgfbr2 flox/flox MEFs, in which Tgf-beta receptor II was deleted. Interestingly, the p38 MAPK inhibitor SB203580 partially impeded Arf induction, showing cross-talk with Smad signals. Chromatin immunoprecipitation revealed that Tgf-beta2 induced the binding of Smad2/3 and RNA Polymerase II to genomic DNA proximal to Arf exon 1 beta. Increased promoter binding preceded the progressively induced Arf mRNA and primary Arf transcripts at 24 and 48 hours, indicating that the induction was due to increased transcription. In summary, our results indicate that Tgf-beta2 induces the Arf promoter via a direct Smad-dependent signaling pathway. This study provides the first evidence that Arf expression is coupled to cell extrinsic signals. We are currently exploring this pathway in other type of cells and testing whether Arf induction by oncogenes also depends on Tgf-beta signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1097.