[Alteration of Microparticle Levels in Early Complications During Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the alteration of microparticles (MP) in the recipients following hematopoietic stem cell transplantation (HSCT) and its significance, and to search the early diagnostic indicators of thrombotic complications after transplantation. METHODS: According to the occurrence of transplantation-associated complications, 94 allo-HSCT patients were divided into 4 groups: thrombotic group (VOD n = 7, TMA n = 2), acute graft-versus-host disease (aGVHD) group (n = 27), infection group (n = 41) and non-complication group (n = 17). Alterations of serum concentration of tissue factor positive microparticles (TF(+) MP) and endothelial microparticles (EMP) were analyzed by flow cytometry during the process of conditioning treatment and the early stage after transplantation. The relation of these 2 kinds of MP with complications was analysed. RESULTS: (1) The levels of TF(+) MP and EMP of patients undogoing allo-HSCT before conditioning treatment were obviously higher than those in normal controls, and showed some elevation during different times, but there was no significant statistical difference. Although the levels of TF(+) MP and EMP at the end of conditioning treatment were some higher than those before conditioning treatment, but there was no statistical difference between them. (2)The levels of TF(+) MP and EMP in thrombotic group were obviously higher than those in aGVHD group and infection group (P < 0.05). (3)The levels of TF(+) MP and EMP in thrombotic group at different times were significant differences from those in other groups (P < 0.05), and the levels of TF(+) MP and EMP were no significant difference from those in non-complication group. CONCLUSION: The increase of the TF(+) MP and EMP levels may be associated with occurrence of thrombosis after transplantation, indicating occurrence of the thrombotic complications, like hepatic vein occulusive disease (HVOD). The dynamically monitoring levels of TF(+) MP and EMP contributes to early discovery of thrombotic complications.

PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.

PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes.

PTEN C-terminal deletion causes genomic instability and tumor development.

Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here, we describe a knockin mouse model of a nonsense mutation that results in the deletion of the entire Pten C-terminal region, referred to as Pten(ΔC). Mice heterozygous for Pten(ΔC) develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C-terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing the initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C terminus in the maintenance of genomic stability and suppression of tumorigenesis.

[Expression characteristics of differentiation antigens on granulocytes in patients with megaloblastic anemia].

This study was aimed to explore the change characteristics of cell differentiation antigen (CD) on bone marrow (BM) granulocytes in patients,with megaloblastic anemia (MA). In combination with BM cell morphology, hemogram, level of blood serum folic acid, level of Vit B(12), cell genetics and biological examination data, the BM granulocytes differentiation antigens in 13 patients with MA were detected by flow cyto metry and analyzed retrospectively, in order to summarize the variation characteristics of CD13, CD33 and CD15 expressed on myeloid cells in patient with MA, including forward scatter light (FSC) and side scatter light (SSC) signal intensity, then these findings were compared with that in normal healthy persons. The results showed that the expression rates of CD13, CD15 and CD33 on granulocytic in patients with MA and normal healthy persons were (44.53 ± 16)%, (96.16 ± 2.67)%, (80.81 ± 14.71)% and (62.33 ± 11.02)%, (99.53 ± 0.46)%, (70.00 ± 7.81)% respectively, in which the expression rate of CD13 and CD15 in patients with MA decreased (P < 0.01), while the expression rate of CD33 increased (P < 0.01). The mean fluorescence intensity (MFI) of CD13, CD15, CD33, SSC and FSC in MA patients and normal healthy persons were 3.39 ± 1.41, 14.29 ± 6.59, 1.95 ± 0.94, 478.78 ± 70.43, 633.46 ± 75.53 and 5.12 ± 1.15, 20.67 ± 5.13, 1.04 ± 0.17, 332.00 ± 38.16, 537.00 ± 16.70 respectively, in which the MFI of CD13 and CD15 on granulocytes in MA patients decreased (P < 0.01),while the MFI of FSC,SSC and CD33 increased (P < 0.01 and P < 0.05). It is concluded that not only the morphology of BM granulocytes in patents with MA shows dysmaturity, but the expressing feature of differentiation antigens on BM granulocytes in MA patients also displays dysmaturity.These findings will contribute to the clinical diagnosis of MA patients.

[Detection of tissue factor-positive microparticles and its clinical significance in the haemostatic disorder].

Objective of this study was to detect the level of tissue factor-positive microparticles (TF(+)MP) by flow cytometry (FCM) and to analyze its clinical significance in the haemostatic disorder. TF(+) MP was detected by FCM using antibody CD142-PE in 25 cases of acute promyelocytic leukemia (APL), 20 cases of hemostatic diseases and 20 healthy adults as controls. The differences of TF(+) MP between various groups were determined. The results showed that the level of TF(+) MP in the patients with thrombotic complications was significantly higher than that in the healthy adults (P < 0.05). The TF(+) MP level was higher in the patient with APL than that in the healthy adults, especially in course before therapy (P < 0.01), but the difference was not statistically significant in the patient with APL after therapy and the healthy adults. Among these patient with APL, the level of TF(+) MP in the 18 patients who complicated with disseminated intravascular coagulation (DIC) was also higher than that in the healthy adults (P < 0.05), but the level of TF(+) MP in the other 7 patients who did not complicate with DIC was similar before and after treatment. It is concluded that the method of TF(+) MP detection by FCM is feasible and simple, it is useful for the diagnosis of thrombotic disorder, and helps evaluation for the prognosis of APL patient.

CREB is a novel nuclear target of PTEN phosphatase.

PTEN phosphatase is a potent tumor suppressor that regulates multiple cellular functions. In the cytoplasm, PTEN dephosphorylates its primary lipid substrate, phosphatidylinositol 3,4,5-trisphosphate, to antagonize the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. It has also become increasingly evident that PTEN functions in the nucleus and may play an important part in transcription regulation, but its nuclear targets remain elusive. In this report, we demonstrate the transcription factor cyclic AMP response element-binding protein (CREB) is a protein target of PTEN phosphatase and that PTEN deficiency leads to CREB phosphorylation independent of the PI3K/AKT pathway. Using confocal immunofluorescence and reciprocal immunoprecipitation, we further show that PTEN colocalizes with CREB and physically interacts with CREB. Moreover, we use both in vitro and in vivo experiments to show PTEN can dephosphorylate CREB in a phosphatase-dependent manner, suggesting that CREB is a substrate of PTEN nuclear phosphatase. Loss of Pten results in an elevated RNA level of multiple CREB transcriptional targets and increased cell proliferation, which can be reversed by a nonphosphorylatable CREB mutant or knockdown of CREB. These data reveal a mechanism for PTEN modulation of CREB-mediated gene transcription and cell growth. Our study thus characterizes PTEN as a nuclear phophatase of a transcription factor and identifies CREB as a novel protein target of PTEN phosphatase, which contributes to better understanding of PTEN function in the nucleus.

[Detecting minimal residual disease status in allogeneic hematopoietic stem cell transplantation of patients with high-risk acute leukemia].

OBJECTIVE: To explore the relationship between minimal residual disease (MRD) and the outcome of patients with high-risk acute leukemia (AL) undergoing allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: By 4/5-color multi-parameter flow cytometry (MFC, CD45/SSC gating) for detecting MRD at pre-(day-30) and post-transplant (day +30, +60, +100, 6 months, 9 months and 12 months), the investigators retrospectively analyzed the MRD levels and the prognosis of 90 high-risk patients. According to the MRD cutoff value of 0.1%, the low-level and high-level groups were defined. In the high-level group, the patients were divided into two sub groups according to the subsequent treatment (intervention therapy group and non-intervention therapy group). RESULTS: MRD pre-transplant had no predictive value for the clinical outcome. The patients with high levels of MRD post-transplant (+60 d and +100 d) showed higher relapse rates than those of the low-level group. In addition, regarding MRD +100 d post-transplant, differences were significant among 3 groups (high-level MRD and intervention therapy group, high-level MRD and non-intervention therapy group and low-level MRD group) including 1-year relapse-free survival (RFS) (100% vs 60.87% vs 91.30%, P < 0.05) and 3-year RFS (85.71% vs 44.72% vs 68.48%, P < 0.05). The median time from first high level MRD detected to clinical relapse was 2.5 (1 - 26) months. In the high level MRD group (+100 d post-transplant), 7 of 30 patients received intervention therapy without relapse. However another 23 patients had no intervention treatment and 11 of them relapsed latter (P < 0.05). CONCLUSION: The MFC-based quantification of MRD post-transplant reveals important prognostic information in patients with high-risk AL. MRD check point at day +100 (cutoff: 0.1%) may discriminate different risk populations. Those patients with MRD levels ≥ 0.1% should receive early intervention at an early stage and a low tumor burden so as to reduce the relapse rate and boost survival.

[Expression of endothelial protein C receptor in tumor cell lines and It's significance].

AIM: To detect the role of activated protein C(APC) on proliferation of endothelial cell and investigate the expression of endothelial protein C receptor( EPCR) in variety of tumor cell lines. METHODS: The effect of APC on endotheliocyte proliferation was determined by MTT colorimetry. IL-6 and IL-8 in supernatant were measured by ELISA. Expression of EPCR were measured by semi-quantitative RT-PCR. RESULTS: APC can increase the proliferation of EC significantly. EPCR gene was found in 91.7% of solid tumors and 66.7% of hematopoietic malignancies. CONCLUSION: APC can stimulate the proliferation of endothelial cell. High expression of EPCR in tumor cell lines provides a potential biological marker for malignancies.

[Influence of arsenic trioxide and daunorubicin on the expression of annexin II and fibrinolytic activity in NB4 cells].

OBJECTIVE: To study the expression of annexin II (AnnII) and the fibrinolytic activity in NB4 cells and their alterations in the presence of arsenic trioxide (ATO) and daunorubicin (DNR). METHODS: Leukemia cell line NB4 was treated with ATO or DNR for 24 ∼ 72 h. Cell surface expression of AnnII and its mRNA were analysed by flow cytometry and real time PCR, respectively, the fibrinolytic activity by chromogenic assay. RESULTS: Compared with other acute leukemia cell lines, the expression of AnnII on untreated NB4 cells was relatively higher. The AnnII positive cell rates on NB4, HL-60, K562, and A3 cells were (94.5 ± 1.6)%, (40.1 ± 2.1)%, (36.3 ± 1.5)% and (11.8 ± 2.5)%, respectively. The fibrinolytic activity of NB4 cells was the greatest with a A value of 0.68 ± 0.02. The fibrinolytic activity of NB4 cells was obviously decreased by ATO, DNR or monoclonal antibody against AnnII, being decreased by 60.4%, 35.8% and 26.0% of the pretreatment level, respectively. The expressions of AnnII and its mRNA in NB4 cells were decreased dramatically after ATO and DNR treated for 48 h. Annexin II positive cells rate were (55.46 ± 4.72)% and (27.00 ± 6.18)%, respectively. CONCLUSION: NB4 cells have strong ability to enhance the catalytic efficiency of the t-PA-dependent plasminogen activation and AnnII on the cell membrane contributes to this activity. Its high fibrinolytic activity can be corrected by ATO and DNR through down-regulating AnnII.

[Clinical study on the fibrinolytic activity in patients with acute promyelocytic leukemia].

OBJECTIVE: To study the fibrinolytic activity in patients with acute promyelocytic leukemia (APL) and its alteration in all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) treatment. METHODS: Plasma fibrinogen concentration was determined with the conventional method, and the levels of fibrin degradation products (FDP) and D-dimer were quantified with ELISA. Plasminogen was measured by chromogenic assay. Cell surface expression of Annexin II and u-PAR and their mRNA levels were measured by flow cytometry and real time-PCR, respectively. RESULTS: The levels of FDP and D-dimer in APL were remarkably higher in APL patients than that in normal controls, while fibrinogen and plasminogen were lower. Both Annexin II and u-PAR were highly expressed on APL cells, which declined after treatment with ATRA and/or ATO, but remained higher than those on normal bone marrow mononuclear cells. CONCLUSION: Abnormally high levels of Annexin II and u-PAR expression on APL cells may contribute to the increased production of plasmin, leading to primary hyperfibrinolysis in APL. ATRA and ATO therapy induces down-regulation of Annexin II and u-PAR expression, which may be contribute, at least in part, to the relief of the hemorrhagic complications in APL.

[Effect of protein kinase C and calcium on the thrombin receptors activation].

OBJECTIVE: To compare the effects of protein kinase C (PKC) and calcium (Ca2+) on platelet aggregation and platelet membrane surface GP I b expression in thrombin receptors activation, and investigate the role of Gq signal transduction pathway in such activation. METHODS: Peptide SFLLRN (PARI-AP) and AYPGKF (PAR4-AP) were used to stimulate platelet, and the effects of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator) on the platelet aggregation and GP I b were analyzed. RESULTS: Either 25 micromol/L PAR1 or 250 micromol/L PAR4 peptide could induce absolute platelet aggregation with a reversible internalization of GP I b. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA while the morphological change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreased GP I b centralization upon PAR1 stimulation [(87.00 +/- 0.04)% and (73.00 +/- 0.08)%, respectively at 1, 2 min, P<0.05], albeit it blocked the internalization of GP I b in PAR4 activation [(44.00 +/- 0.01)% and (46.00 +/- 0.05)%, respectively at 10, 30 min, P <0.05]. Meanwhile, GP I b internalization was blocked by BAPTA in both peptides [(94.00 +/- 0.08)% and (95.00 +/- 0.00)% at 1 min, (92.00 +/- 0.02)% and (94.00 +/- 0.01)% at 2 min, (91.00 +/- 0.02)% and (91.00 +/- 0.02)% at 5 min, (90.00 +/- 0.04)% and (87.00 +/- 0.03)% at 10 min, respectively, P <0.05]. CONCLUSION: PKC and calcium play an important role in thrombin receptor activation. Calcium is closely correlated with such activation, being similar in the two PARs signal pathways. PK C promotes GP I b centralization in PAR1 pathway and accelerates GP I b return to membrane surface in PAR4 pathway.

A molecular link between E2F-1 and the MAPK cascade.

Transcription factor E2F-1 mediates apoptosis and suppresses tumorigenesis. The mechanisms by which E2F-1 functions in these processes are largely unclear. We report here that E2F-1 acts as a transcriptional regulator of MKP-2 (MAPK phosphatase-2), a dual specificity protein phosphatase (DUSP4) with stringent substrate specificity for MAPKs. We show that E2F-1 is required for the cellular apoptotic response to oxidative damage. MKP-2 is greatly increased following oxidative stress, and E2F-1 is necessary for that induction. We found that E2F-1 is physically associated with the MKP-2 promoter and can transactivate the promoter of the MKP-2 gene. Specifically, E2F-1 binds to a perfect palindromic motif in the MKP-2 promoter. Finally, we show that this E2F-1/MKP-2 pathway mediates apoptosis under oxidative stress and that MKP-2 suppresses tumor formation in nude mice. Our findings demonstrate that E2F-1 is a transcriptional activator of MKP-2 and that MKP-2 is an essential cell death mediator in the E2F-1 pathway. Characterization of MKP-2 as a cell death mediator may lead to the development of new strategies for cancer treatment.

Essential role for nuclear PTEN in maintaining chromosomal integrity.

A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. We report a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). We show that PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. Our results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. We propose that PTEN acts as a guardian of genome integrity.

[Effect of anti-Helicobacter pylori ureB monoclonal antibody on platelet aggregation and activation, and its mechanism study].

OBJECTIVES: To study the effect of monoclonal antibody (McAb) against helicobacter pylori (Hp) ureB, 1F11 on platelet aggregation and activation, and its mechanism. METHODS: The relativity between human platelet glycoproteins (GPs) and Hp ureB was identified by Western blot and FCM. Platelet aggregation was measured by turbidimetry, and P-selectin and TXB2 assay by ELISA. RESULTS: 1F11 could bind to platelet GPIIIa, and ADP-induced platelet aggregation was inhibited by 1F11 in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 induced by ADP. The FCM results show that the positive rates of platelet binding to FITC-SZ21 was decreased from 99.5% to 77.4% after addition of 1F11. CONCLUSION: McAb against Hp ureB 1F11 inhibits platelet aggregation through binding to platelet GPIIIa but does not block platelet activation. There might be crossed-epitopes on Hp ureB and platelet GPIIIa, and Hp infection might be involved in ITP immunopathology.

C-jun N-terminal kinase mediates tumor necrosis factor-alpha suppression of differentiation in myoblasts.

The stress kinase c-jun N-terminal kinase (JNK) was recently shown to be involved in the pathophysiology of major inflammatory conditions, including Alzheimer's disease, stroke, obesity, and type II diabetes. However, the role of JNK in regulating inflammatory events in skeletal muscle is only beginning to be explored. IGF-I is the major hormone that promotes muscle growth and development. Here we used a novel, JNK interacting protein (JIP)-derived JNK peptide inhibitor to establish that JNK suppresses the biological activity of IGF-I in skeletal muscle progenitor cells. In these myoblasts, TNFalpha and its downstream receptor substrates, neutral-sphingomyelinase (N-SMase) and N-acetyl-d-sphingosine (C2-ceramide), induce JNK kinase activity in a time-dependent manner. Consistent with these results, TNFalpha induces JNK binding to insulin receptor substrate 1 (IRS-1) but is unable to inhibit IGF-I-induced IRS-1 tyrosine phosphorylation in myoblasts that are treated with the JNK peptide inhibitor. More importantly, JNK activation induced by TNFalpha, C2-ceramide, and N-SMase is associated with reduced expression of the critical muscle transcription factor myogenin as well as the differentiation marker myosin heavy chain (MHC). The JNK peptide inhibitor, but not the control peptide, completely reverses this inhibition of both myogenin and MHC. In the absence of IGF-I, TNFalpha, C2-ceramide, N-SMase and the JNK inhibitor are inactive, as shown by their inability to affect IRS tyrosine phosphorylation and protein expression of myogenin and MHC. These results establish that the resistance of muscle progenitor cells to IGF-I, which is caused by inflammatory stimuli, is mediated by the JNK stress kinase pathway.

Mitogen-activated protein kinase phosphatase 2: a novel transcription target of p53 in apoptosis.

The p53 tumor suppressor plays critical roles in diverse cellular responses such as cell cycle arrest, senescence, and apoptosis through transcriptional control of its target genes. Identification and characterization of new p53 target genes will advance our understanding of how p53 exerts its multiple regulatory functions. In this article, we show that mitogen-activated protein kinase phosphatase 2 (MKP2) is a novel transcription target of p53 in mediating apoptosis. Moreover, we identify a 10-bp perfect palindrome motif (CTGGCGCCAG) in the MKP2 promoter as a new binding site for p53 to activate the MKP2 gene. This GC-rich palindrome is completely different from the consensus p53 binding sequence. Induction of MKP2 is highly responsive to oxidative stress in a p53-dependent manner. Interestingly, the p53-dependent induction of MKP2 is prominent only in the cellular response to stimuli leading to apoptosis but not to cell cycle arrest. In response to oxidative stress, MKP2 is not only required for p53-mediated apoptosis, but ectopic MKP2 expression can also enhance apoptotic responses even independent of p53. These data suggest that p53 regulates distinct genes via different binding mechanisms and that MKP2 is an essential target of p53 in signaling apoptosis.

[Role of adenosine diphosphate in the course of thrombin signal transmission].

OBJECTIVE: To study the effects adenosine diphosphate (ADP) on platelet aggregation and expression of glycoprotein (GP) on the surface of platelet membrane after activation of thrombin receptors, so as to investigate its role in thrombin signal transmission. METHODS: Peripheral blood samples were collected from from 10 healthy volunteers. Platelets were extracted. The thrombin receptor activating peptides (TRAP), protease-activated receptor 1 activated peptide (PAR1-AP, SFLLRN, 25 micromol/L) and PAR4-AP (AYPGKF, 250 micromol/L) were added into the suspension of platelets respectively to induce platelet aggregation. In apyrase inhibition test apyrase II was added into the suspension of platelets for 2 hours and then PAR1-AP or PAR4-AP was added respectively to observe the the expression of GPIb and P-selectin with flow cytometry. RESULTS: Either PAR1 and PAR4 induced platelet aggregation. After apyrase II stimulation the PAR4-AP induced platelet aggregation was not influenced and PAR1-AP induced platelet aggregation was partially inhibited with a reversible aggregation curve. Stimulated by PAR1-AP and PAR4-AP the GPIb decreased firstly and then gradually returned to normal. Apyrase VII have not significant influence on the GPIb expression, but accelerated the return of GPIb to the platelet surface after PAR1 stimulation so that the lowest point was accelerated to 2 min, compared to that of the control group (5 min) and there were significantly differences 10 and 30 min later between these 2 groups (all P < 0.05). The P-selectin expression was remarkably increased 2 min after the PAR1-AP and PAR4-AP induction and peaked 2 min later. Apyrase VII did not significantly influence the P-selectin expression in these 2 activation ways. CONCLUSION: ADP plays an important role in the thrombin signal transmission, especially in the PAR1 pathway.

Proinflammatory cytokine impairment of insulin-like growth factor I-induced protein synthesis in skeletal muscle myoblasts requires ceramide.

GH and IGF-I control over 80% of postnatal growth. We recently established that TNFalpha impairs the ability of IGF-I to increase protein synthesis and promote expression of myogenin in myoblasts. Here we extend these results by showing that ceramide, a second messenger in both TNFalpha and IL-1beta receptor signaling pathways, is a key downstream sphingosine-based lipid that leads to IGF-I resistance. A cell-permeable ceramide analog, C2-ceramide, inhibits IGF-I-induced protein synthesis by 65% and blocks the ability of IGF-I to increase expression of two key myogenic factors, myogenin and MyoD. Identical results were obtained with both TNFalpha and IL-1beta (1 ng/ml). Consistent with these data, neutral sphingomyelinase (N-SMase), an enzyme that catalyzes formation of ceramide from sphingomyelin, blocks IGF-I-induced protein synthesis and expression of both myogenin and MyoD. The possibility that cytokine-induced ceramide production is required for disruption of IGF-I biologic activity was confirmed by treating C2C12 myoblasts with inhibitors of all three ceramide-generating pathways. A N-SMase inhibitor, glutathione, as well as an acidic sphingomyelinase (A-SMase) inhibitor, D609, reverse the cytokine inhibition of IGF-I-induced protein synthesis by 80% and 45%, respectively. Likewise, an inhibitor of de novo ceramide synthesis, FB1, causes a 50% inhibition. Similarly, all three inhibitors significantly impair the ability of both TNFalpha and IL-1beta to suppress IGF-I-driven expression of myogenin. These experiments establish that ceramide, derived both from sphingomyelin and de novo synthesis, is a key intermediate by which proinflammatory cytokines impair the ability of IGF-I to promote protein synthesis and expression of critical muscle-specific transcription factors.

IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells.

Cell cycle aberrations occurring at the G(1)/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1beta inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G(0)/G(1) arrest. Notably, IL-1beta suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1beta reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1beta has no effect. IL-1beta induces p21(Cip1/Waf1), which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1beta; stimulation by growth factors such as IGF-I promotes G(1)-S phase progression, whereas IL-1beta antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.

IL-1beta impairs insulin-like growth factor i-induced differentiation and downstream activation signals of the insulin-like growth factor i receptor in myoblasts.

Proinflammatory cytokines are elevated in disorders characterized by muscle wasting and weakness, such as inflammatory myopathies and AIDS wasting. We recently demonstrated that TNF-alpha impairs the ability of insulin-like growth factor (IGF)-I to promote protein synthesis in muscle precursor cells. In this study we extend these findings by showing that low concentrations of IL-1beta impair IGF-I-dependent differentiation of myoblasts, as assessed by expression of the muscle specific protein, myosin heavy chain. In the absence of exogenous IGF-I, IL-1beta (1 ng/ml) did not impair muscle cell development. However, in the presence of IGF-I, 100-fold lower concentrations of IL-1beta (0.01 ng/ml) significantly suppressed myoblast differentiation, protein synthesis, and myogenin expression. Increasing IL-1beta to 1 ng/ml completely blocked the anabolic actions of IGF-I in murine C(2)C(12) myoblasts. Similarly, IL-1beta inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. IL-1beta impaired the actions of IGF-I at a point distal to the IGF receptor, and this was not due to IL-1beta-induced cell death. Instead, IL-1beta inhibited the ability of IGF-I to phosphorylate tyrosine residues on both of its downstream docking proteins, insulin receptor substrate 1 and insulin receptor substrate 2. These data establish that physiological concentrations of IL-1beta block the ability of IGF-I to promote protein synthesis, leading to reduced expression of the myogenic transcription factor, myogenin, and the subsequent development of more mature differentiated cells that express myosin heavy chain. Collectively, the results are consistent with the notion that very low concentrations of IL-1beta significantly impair myogenesis, but they are unable to do so in the absence of the growth factor IGF-I.