SIMPL enhancement of tumor necrosis factor-α dependent p65-MED1 complex formation is required for mammalian hematopoietic stem and progenitor cell function.

Significant insight into the signaling pathways leading to activation of the Rel transcription factor family, collectively termed NF-κB, has been gained. Less well understood is how subsets of NF-κB-dependent genes are regulated in a signal specific manner. The SIMPL protein (signaling molecule that interacts with mouse pelle-like kinase) is required for full Tumor Necrosis Factor-α (TNFα) induced NF-κB activity. We show that SIMPL is required for steady-state hematopoiesis and the expression of a subset of TNFα induced genes whose products regulate hematopoietic cell activity. To gain insight into the mechanism through which SIMPL modulates gene expression we focused on the Tnf gene, an immune response regulator required for steady-state hematopoiesis. In response to TNFα SIMPL localizes to the Tnf gene promoter where it modulates the initiation of Tnf gene transcription. SIMPL binding partners identified by mass spectrometry include proteins involved in transcription and the interaction between SIMPL and MED1 was characterized in more detail. In response to TNFα, SIMPL is found in p65-MED1 complexes where SIMPL enhances p65/MED1/SIMPL complex formation. Together our results indicate that SIMPL functions as a TNFα-dependent p65 co-activator by facilitating the recruitment of MED1 to p65 containing transcriptional complexes to control the expression of a subset of TNFα-induced genes.

Loss of SIMPL compromises TNF-alpha-dependent survival of hematopoietic progenitors.

OBJECTIVE: Emerging work has revealed an integral role of the tumor necrosis factor-alpha (TNF-alpha) nuclear factor (NF)-kappaB pathway in the regulation of hematopoiesis. TNF-alpha inhibition of hematopoietic stem/progenitor cell growth involves type I TNF-alpha receptor (TNF-RI) and type II TNF-alpha receptor (TNF-RII). However, the role of TNF-RI vs TNF-RII in mediating this response is less clear. Full induction of NF-kappaB-dependent gene expression through TNF-RI requires the transcriptional coactivator SIMPL (substrate that interacts with mouse pelle-like kinase). To address the role of SIMPL in TNF-alpha-dependent signaling in hematopoiesis, endothelial cells and hematopoietic progenitors expressing SIMPL short hairpin RNA were characterized. MATERIAL AND METHODS: In vitro gene expression and progenitor assays employing SIMPL short hairpin RNA were used to examine the requirement for SIMPL in TNF-alpha-dependent effects upon cytokine gene expression and hematopoietic progenitor cell growth. Competitive repopulation studies were used to extend these studies in vivo. RESULTS: SIMPL is required for full TNF-RI-dependent expression of NF-kappaB-controlled cytokines in endothelial cells. Hematopoietic progenitor cell expansion is not affected if progenitors lacked SIMPL or if progenitors are treated with human TNF-alpha, which signals through TNF-RI. In the absence of SIMPL, human TNF-alpha leads to a dramatic decrease in progenitor cell expansion that is not due to apoptosis. Loss of SIMPL does not affect the activity of transforming growth factor-beta1 and interferon-gamma, other known suppressors of hematopoiesis. CONCLUSIONS: Suppression of myeloid progenitor cell expansion requires signaling through TNF-RI and TNF-RII. Signals transduced through the TNF-alpha-TNF-RI-SIMPL pathway support hematopoietic progenitor cell survival, growth and differentiation.

Transient membrane recruitment of IRAK-1 in response to LPS and IL-1beta requires TNF R1.

The transcription factor NF-kappaB is an essential regulator of the innate immune response that functions as the first line of defense against infections. Activation of the innate immune response by bacterial lipopolysaccharide (LPS) triggers production of tumor necrosis factor-alpha (TNF-alpha) followed by interleukin-1 (IL-1). The IL-1 receptor associated kinase-1 (IRAK-1) is an integral component of the LPS, TNF-alpha, and IL-1 signaling pathways that regulate NF-kappaB. Thus we hypothesized that IRAK-1 coordinates cellular NF-kappaB responses to LPS, TNF-alpha, and IL-1. In contrast to TNF-alpha where IRAK-1 subcellular localization does not change, treatment with LPS or IL-1 leads to a loss in cytoplasmic IRAK-1 with a coordinate increase in plasma membrane associated modified IRAK-1. In fibroblasts lacking the type 1 TNF-alpha receptor (TNF R1), IRAK-1 turnover is altered and modification of IRAK-1 in the plasma membrane is decreased in response to LPS and IL-1, respectively. When NF-kappaB controlled gene expression is measured, fibroblasts lacking TNF R1 are hyperresponsive to LPS, whereas a more variable response to IL-1 is seen. Further analysis of the LPS response revealed that plasma membrane-associated IRAK-1 is found in Toll 4, IL-1, and TNF R1-containing complexes. The data presented herein suggest a model whereby the TNF R1-IRAK-1 interaction integrates the cellular response to LPS, TNF-alpha, and IL-1, culminating in a cell poised to activate TNF-alpha-dependent NF-kappaB controlled gene expression. In the absence of TNF R1-dependent events, exposure to LPS or IL-1 leads to hyperactivation of the inflammatory response.

Tumor Necrosis Factor-alpha- and interleukin-1-induced cellular responses: coupling proteomic and genomic information.

The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFalpha) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFalpha- and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFalpha and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFalpha and IL-1 regulate different processes. A large-scale proteomic analysis of TNFalpha- and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFalpha and IL-1. When combined with genomic studies, our results indicate that TNFalpha, but not IL-1, mediates cell cycle arrest.

Phosphorylation of SIMPL modulates RelA-associated NF-kappaB-dependent transcription.

Epidemiological data have implicated perturbations in the regulation of NF-kappaB activity to diseases that affect a large number of Americans today. Specifically, chronic activation of genes involved in the inflammatory response is associated with the progression of and complications in diabetes, arthritis, atherosclerosis, and cancer. Insight into the mechanisms governing the regulation of NF-kappaB transcriptional activity will provide the molecular link between NF-kappaB and these pathological states. SIMPL (signaling molecule that associates with mouse Pelle-like kinase) is a component of a signaling pathway through which tumor necrosis factor-alpha (TNF-alpha) induces NF-kappaB-controlled gene transcription. SIMPL interacts with the nuclear pool of the NF-kappaB subunit, p65, in a TNF-alpha-dependent manner to enhance p65-dependent gene transcription. How SIMPL activity is regulated is unknown. Under basal as well as TNF-alpha-stimulated conditions, SIMPL phosphopeptides were identified. SIMPL mutants lacking sites that are phosphorylated under basal conditions diminished p65 transactivation activity but had no effect on SIMPL nuclear localization. SIMPL mutants lacking sites of TNF-alpha-enhanced phosphorylation impaired nuclear localization and prevented TNF-alpha-induced p65 transactivation activity. Together, these studies reveal that phosphorylation of the SIMPL protein plays a critical role in SIMPL regulation by affecting both SIMPL subcellular localization and the p65 coactivator function of SIMPL.

Alpha-lipoic acid modulates ovarian surface epithelial cell growth.

OBJECTIVE: The intracellular redox state plays an important role in controlling inflammation. Clinical and laboratory data suggest that inflammation can lead to tumor progression. We hypothesized that restoring intracellular redox control would inhibit inflammation and subsequently tumor progression. Our studies were designed to investigate the effect of alpha-lipoic acid (ALA), a naturally occurring antioxidant, on a key inflammatory signaling pathway and cell proliferation in normal and tumorigenic ovarian surface epithelial cells. METHODS: Normal and tumorigenic ovarian surface epithelial cells were isolated as described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585. [1]]. The effect of ALA on cellular function was measured in cell proliferation and apoptosis assays. p27(kip1) protein levels were measured by Western analysis. Activation of NF-kappaB dependent transcription was assessed in cell cultures transiently transfected with NF-kappaB controlled reporter constructs. RESULTS: Our results reveal that ALA selectively inhibits the growth of tumorigenic as compared to non-tumorigenic ovarian surface epithelial cells. The growth inhibitory effect of ALA is not due to induction of apoptosis but instead is associated with an increase in the half-life of the cyclin-dependent kinase inhibitor, p27(kip1). In parallel to the growth inhibitory effect, ALA also affects a key inflammatory signaling pathway by inhibiting TNFalpha-induced NF-kappaB signaling activity. CONCLUSIONS: Our studies are the first to show that ALA treatment has a growth inhibitory effect on malignant surface epithelial cells of ovarian origin. We have also confirmed the reproducibility of the immunocompetent mouse ovarian cancer model originally described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585].

The disordered amino-terminus of SIMPL interacts with members of the 70-kDa heat-shock protein family.

The p65 coactivator SIMPL is a small protein that lacks any conserved domains of known function. To better understand regulation of SIMPL activity, we sought to identify novel SIMPL interacting proteins using mass spectrometry analysis of SIMPL containing complexes. Two members of the 70-kDa heat-shock protein family, Hsp70 and Hsc70, were identified as SIMPL binding proteins. Subsequent immunocomplexing assays confirmed this interaction and demonstrated that the amino-terminus of SIMPL is required for this interaction. Using a combination of amino acid composition analysis, PONDR VL-XT prediction, charge-hydropathy plots, and cumulative distribution functions, the amino-terminal region of both mouse and human SIMPL proteins was predicted to be intrinsically disordered. These data, taken together, suggest that Hsp70/Hsc70 bind the intrinsically disordered amino-terminal region of SIMPL to stabilize the protein and thereby regulate its activity. Understanding the regulation of SIMPL through its interaction with Hsp70/Hsc70 may serve as a novel means of modulating tumor necrosis factor alpha signaling.

Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by Mouse Pelle-like kinase/interleukin-1 receptor-associated kinase: cross-talk between inflammatory signaling and insulin signaling that may contribute to insulin resistance.

Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-alpha-treated cells. In NIH-3T3(IR) cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-alpha. Using mass spectrometry, we identified Ser(24) in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser(24)) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-alpha or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser(24), we found that interleukin-1 or TNF-alpha treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser(24). We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-alpha-regulated phosphorylation at Ser(24) in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.

Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL.

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression.

Transcriptional targeting in ovarian cancer cells using the human epididymis protein 4 promoter.

OBJECTIVE: Limitations of current ovarian cancer gene therapies include lack of specificity and transduction of normal tissues. One strategy toward overcoming these limitations is to direct gene therapy specifically to ovarian cancer cells by using tissue- and tumor-specific promoters. The whey-acidic protein human epididymis protein 4 (HE4) is frequently overexpressed in ovarian cancer, suggesting that the HE4 promoter is highly transcriptionally active in the disease. The objective of this study was to isolate the HE4 promoter and examine its ability to selectively activate reporter gene expression in an ovarian cancer-specific manner. METHODS: To investigate transcriptional targeting in ovarian cancer gene therapy, we isolated a region of the HE4 promoter from -530 to +122 (pHE4-652; relative to the ATG start site of HE4) and placed it upstream of a luciferase reporter gene plasmid to generate pHE4-652-luc. The activity of the pHE4-652-luc reporter construct was characterized in transient transfection assays in a panel of epithelial ovarian cancer cell lines (SKOV-3, SKOV-3x, CP70, HeyC2, A2780, A2780CP, OVCAR-3), non-ovarian tumor cell lines, and primary cultures of normal cells. The activity of two other candidate gene therapy promoters, human telomerase reverse transcriptase (hTERT) and OSP1, was also characterized in these cell lines. RESULTS: The HE4 promoter was active in 5/7 ovarian cancer cell lines with the range of activity spanning 0.06- to 3-fold that observed for a positive control, cotransfected reporter construct (SV-40-luc). Minimal pHE4-652 promoter activity, defined as < or =5% of the activity detected with the SV-40-luc construct, was observed in the non-ovarian tumor cell lines and normal cells. The hTERT and the OSP1 promoters were active in the ovarian cancer lines. hTERT activity was highest in the CP70 cell line, and OSP1 activity was highest in the SKOV-3x cell line. Modest OSP1 and hTERT promoter activity was observed in normal cell lines and in selected non-ovarian cancer cell lines. CONCLUSION: This is the first report using the pHE4-652 promoter to drive specific reporter gene expression in epithelial ovarian cancer cell lines, and we are continuing to develop this promoter for use in transcriptional targeting in ovarian cancer gene therapy.

Cutting edge: mouse pellino-2 modulates IL-1 and lipopolysaccharide signaling.

Pellino is a Drosophila protein originally isolated in a two-hybrid screen for proteins interacting with the serine/threonine kinase, pelle. Although mammalian homologs have been identified in mouse and man, the function of pellino is as yet unknown. In this study, the cloning, expression pattern, and a preliminary characterization of mouse pellino-2 is described. These studies reveal that mouse pellino-2 is expressed during embryogenesis and in a tissue-restricted manner in the adult. IL-1 induces the association of mouse pellino-2 with the mouse pelle-like kinase/IL-1R-associated kinase protein, a mammalian homolog of pelle. Ectopic pellino-2 expression did not result in NF-kappaB activation. However, ectopic expression of a mouse pellino-2 antisense construct inhibited IL-1 or LPS-induced activation of NF-kappaB-dependent IL-8 promoter activity. Our data reveal that mouse pellino-2 is a tissue-restricted component of a signaling pathway that couples the mouse pelle-like kinase/IL-1R-associated kinase protein to IL-1- or LPS-dependent signaling.

Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27(Kip1) inhibits cell growth.

OBJECTIVE: The purpose of this study was to test the hypothesis that the expression of the mutant p27(Kip1) protein enhances cell growth inhibition and is more stable than that of the wild-type p27(Kip1). METHODS: Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27(Kip1). To study the effects of the p27(Kip1) mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27(Kip1), or mutant p27(Kip1) using Fugene 6. The transfected cells were lysed and assayed for luciferase activity 24 h later with a dual-luciferase reporter assay system. To further assess the effects of the p27(Kip1) mutant on cell growth, colony count assays were performed. The experiments were repeated in duplicate and a standard two-tailed Student t test was use to analyze the data. RESULTS: Wild-type p27(Kip1) protein has a half-life of approximately 2 h while the p27(Kip1) mutant has a half-life of greater than 12 h. Furthermore, the p27(Kip1) mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27(Kip1) mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27(Kip1) (P = 0.001). Colony assays revealed that cells expressing the p27(Kip1) mutant had fewer colonies compared to cells expressing the wild-type p27(Kip1) (P = 0.04). CONCLUSIONS: These data are consistent with the hypothesis that the mutated form of p27(Kip1) is more effective in cell growth inhibition than the wild-type p27(Kip1) protein.