Possible role of RKIP in cytotrophoblast migration: immunohistochemical and in vitro studies

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen-activated protein kinases (MAPK), GRK2 and NF-kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre-eclampsia (PE)-complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR-8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR-8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR-8/SV neo cells, suggesting the involvement of GRK2 and NF-kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration.     

Intracellular overexpression of HIV-1 Nef impairs differentiation and maturation of monocytic precursors towards dendritic cells

Nef functions as an immunosuppressive factor critical for HIV-1 replication, survival and development of AIDS following HIV-1 infection. What effects Nef exerts on differentiation and maturation of monocytes towards dendritic cells (DCs) remains greatly controversial. In this study, we used THP-1 (human monocytic leukemia cell line) as monocytic DC precursors to investigate how overexpression of HIV-1 Nef influences the processes of differentiation and maturation of dendritic cells. In striking contrast to negative controls, our results showed that morphological and phenotypical changes (CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR) occurred on recombinant THP-1 expressing HIV-1 Nef (short for Nef) upon co-stimulation of GM-CSF/IL-4 or GM-CSF/IL-4/TNF-α/ionomycin. Moreover, CD4, CCR5, and CXCR4 were also down-regulated on Nef. It might be hypothesized that Nef prevents superinfection and signal transduction in HIV-1 infected monocytes. Collectively, our study demonstrates that long-lasting expression of Nef at high levels indeed retards differentiation and maturation of dendritic cells in terms of phenotype and morphology. We are hopeful that potentially, stable expression of intracellular Nef in vivo may function as a subtle mode to support long-lasting HIV-1 existence.     

Inducer-and cell type-specific regulation of antiapoptotic MCL1 in myeloid leukemia and multiple myeloma cells exposed to differentiation-inducing or microtubule-disrupting agents

The antiapoptotic BCL2 family member MCL1 is rapidly upregulated upon exposure of ML-1 myeloid leukemia cells to either differentiation-inducing phorbol 12′-myristate 13′-acetate (PMA) or chemotherapeutic microtubule disrupting agents (MTDAs). This report examined how signaling for MCL1 upregulation is coupled to these two different phenotypic changes, and tested for upregulation in other hematopoietic cancers. With PMA, ERK stimulated MCL1 mRNA expression and ML-1 cell differentiation, and ERK additionally stabilized expression of the MCL1 protein. However, with MTDAs, transient ERK and ensuing JNK activation contributed to initial MCL1 upregulation and viability-retention, but sustained JNK activation eventually resulted in cell death. MCL1 was upregulated by PMA in THP-1 and U937 myeloid leukemia cells, but by MTDAs only in THP-1 cells. MCL1 expression was constitutively elevated in multiple myeloma cell lines, and was not affected by PMA/ERK or MTDAs. Thus, MCL1 expression level and sensitivity to regulation are important considerations in selecting approaches for targeting this antiapoptotic gene product to kill cancer cells.     

即早基因c-fos在THP-1单核巨噬细胞亚型极化中的差异表达*

目的:探讨即早基因c-fos在THP-1巨噬细胞亚型极化过程中的表达变化。方法:运用PMA刺激诱导THP-1单核细胞极化为巨噬细胞,观察c-fos在单核细胞极化过程中的表达变化;在PMA刺激的基础上,分别运用LPS和IL-4诱导THP-1巨噬细胞向M1及M2亚型极化,实时定量PCR及Western blot技术分析刺激24 h时,细胞亚型标记物CD274、CD86和CD163的表达变化,并动态观察诱导极化过程中,c-fos的表达情况。结果:c-fos在PMA刺激THP-1单核细胞分化为巨噬细胞过程中蛋白和mRNA水平显示上调;LPS诱导THP-1巨噬细胞极化为M1型过程中,c-fos蛋白和mRNA水平表达降低,其特异性标记物在24 h呈现出M1型极化的特点(CD86蛋白表达升高,CD274、CD163蛋白表达降低);IL-4诱导THP-1巨噬细胞极化为M2型过程中,c-fos蛋白和mRNA水平表达升高,其特异性标记物在24 h表现出M2型极化的特点(CD86蛋白表达降低,CD274、CD163蛋白表达升高)。结论:c-fos参与了THP-1单核细胞向巨噬细胞极化的过程,并且可能通过抑制巨噬细胞M1亚型形成,促进巨噬细胞向M2亚型极化的作用参与巨噬细胞的亚型极化及其功能调节中。     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl⁺ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G₁ arrest via p27^Kip1 and p21^Cip1 accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl⁺ progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl⁺ progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.     

MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells

The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.     

The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of “immune coagulation” and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as well as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.     

Triglyceride enhances susceptibility to TNF-α-induced cell death in THP-1 cells

Monocytes and macrophages play a major role in atherosclerosis development. Previously, we found that triglyceride (TG) promoted cell death of PMA-differentiated THP-1 macrophages. In this study, we compared the responsiveness of THP-1 monocytes and PMA-differentiated THP-1 macrophages to TNF-α-induced cell death. We found that, whereas THP-1 monocytes were TNF-α-resistant, THP-1 macrophages were sensitive to TNF-α-induced cell death. THP-1 monocytes treated with TG underwent cell death beginning at 24 h and addition of TNF-α further increased cell death. Based on these observations, we hypothesized that TG-induced differentiation of THP-1 monocytes into THP-1 macrophages, subsequently allowing sensitivity to TNF-α. To determine if TG could induce differentiation of THP-1 monocytes into THP-1 macrophages, we examined the mRNA expression levels of the macrophage-specific markers, CD11b, CD18, CD36 and CD68, by RT-PCR analysis. Our results show that expression of CD11b, CD36 and CD68 increased in TG-treated THP-1 monocytes in a dose- and time-dependent manner; furthermore, TNF-α expression was upregulated in TG-treated THP-1 monocytes. We have concluded that TG induces differentiation of THP-1 monocytes into macrophages concomitant with the production of TNF-α and increased sensitivity to TNF-α-dependent cell death.     

Regulation of progranulin expression in myeloid cells

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.     

Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G_i/o, whereas the SPC-induced calcium influx was dependent on G_q. Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G_i/o and G_q. These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms.     

Differential regulation of the human IFN-gamma receptor expression in Raji and IM9 lymphoblastoid cells versus THP-1 monocytic cells by IFN-gamma and phorbol myristate acetate

Expression of the human (hu) IFN-gamma-R has been studied in Raji and IM9 cells (two B lymphoblastoid cell lines) and in THP-1 cells (a monocytic cell line) with respect to IFN-gamma binding sites, receptor protein and mRNA levels. Although, in these three cell lines, the hu-IFN-gamma-R mRNA was expressed to the same extent, the high affinity receptor was expressed differently both in cell surface receptor binding and amount of receptor protein. Various ligands are able to modulate the expression of their own receptor. We investigated the modulation of the hu-IFN-gamma-R by its ligand. Hu-IFN-gamma induced a rapid and dose-dependent decrease of its cell surface receptor number without alteration of receptor affinity, amounts of receptor protein or hu-IFN-gamma-R mRNA accumulation and stability. Thus, in Raji, IM9, and THP-1 cells, the hu-IFN-gamma had no effect on its receptor gene expression and the cell surface decrease was simply due to ligand blocking and receptor internalization rather than true down-regulation. The second messenger in the hu-IFN-gamma signal transduction pathway is not well characterized, but activation of protein kinase C has been reported in some cases. Therefore, the modulation of the hu-IFN-gamma-R expression by PMA, a potent activator of protein kinase C and a modulator of other receptor expression, has been investigated. In Raji and IM9 cells, PMA had no or few effects on the cell surface receptor number and no detectable effect on the receptor protein or on mRNA levels. In contrast, in THP-1 cells, PMA treatment induced a time and dose-dependent five- to sixfold increase of the cell surface receptors due to a rapid and persistent increase of the hu-IFN-gamma-R gene expression in THP-1 cells was specifically inhibited or reversed by hu-IFN-gamma treatment. The modulation of the hu-IFN-gamma-R expression by PMA in THP-1 cells and by hu-IFN-gamma in PMA-treated THP-1 cells seems associated with their effect on monocyte-macrophage differentiation and/or macrophage activation.     

Interferon-induced protein IFIT4 is associated with systemic lupus erythematosus and promotes differentiation of monocytes into dendritic cell-like cells

Introduction

Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown.

Methods

In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting.

Results

We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls.

Conclusion

Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.     

Expression of surface antigen and mRNA for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family in hematopoietic cells

We characterized the surface antigen and mRNA expression for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family during hematopoietic cell differentiation. The CD11c subunit antigen and mRNA are constitutively expressed in undifferentiated HL-60 promyelocytic leukemia cells, and levels increase markedly with differentiation along the monocyte/macrophage pathway using phorbol myristate acetate. Human monocyte-derived macrophages and human alveolar macrophages express elevated levels of the CD11c subunit antigen and mRNA, indicating that the changes observed in vitro are present in vivo. Dot blot analysis of immature and mature lymphoid and myeloid cells and cell lines demonstrate equivalent levels of CD11c mRNA expression. We conclude that CD11c gene expression is selectively increased during hematopoietic cell differentiation along the monocyte/macrophage pathway.     

Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis, we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently, the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result, the cell proliferation, anchorage-independent growth, cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore, the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation, migration, or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.