PTPN4 negatively regulates CrkI in human cell lines

PTPN4 is a widely expressed non-receptor protein tyrosine phosphatase. Although its overexpression inhibits cell growth, the proteins with which it interacts to regulate cell growth are unknown. In this study, we identified CrkI as a PTPN4-interacting protein using a yeast two-hybrid, and confirmed this interaction using in vitro GST pull-down and co-immunoprecipitation and co-localization assays. We further determined the interactional regions as the SH3 domain of CrkI and the proline-rich region between amino acids 462 and 468 of PTPN4. Notably, overexpression of PTPN4 inhibits CrkI-mediated proliferation and wound healing of HEK293T cells, while knockdown of PTPN4 by siRNA in Hep3B cells enhances CrkI-mediated cell growth and motility. Moreover, our data show that ectopic expression of PTPN4 reduces the phosphorylation level of CrkI in HEK293T cells. These findings suggest that PTPN4 negatively regulates cell proliferation and motility through dephosphorylation of CrkI.     

Modulation of CD157 expression in multi-lineage myeloid differentiation of promyelocytic cell lines

CD157/BST-1 is expressed on mature myeloid cells but not on their precursors in vivo. Also CD38, a homologous gene to CD157, is upregulated in promyelocytic HL-60 cells by the monocyte and granulocyte differentiation-inducing 1alpha,25dihydroxyvitamin D3 (VD3) and all-trans retinoic acid (ATRA), respectively. We have examined whether CD157 expression is upregulated when the promyeloid HL-60 and/or U937 cells are induced to differentiate into mature phenotypes in vitro. VD3 treatment irreversibly upregulated the expression of CD157 in HL-60 cells but not in U937 cells in a time- and concentration-dependent manner when analyzed by flow cytometry, immunoblotting and/or RT-PCR. Different monocyte and granulocyte lineage inducers induced CD157 expression to varying extents while the macrophage differentiation-inducing phorbol 12-myristate 13-acetate (PMA) induced its down-regulation. Time-kinetics of VD3 treatment of HL-60 cells showed that the appearance of CD157 and CD11b (a differentiation marker) antigens were not substantial up to 24 hours but increased subsequently although the appearance of CD38 became significant within 6 hours. Two-color staining of VD3-treated HL-60 cells displayed an apparently linear correlation between CD157 and CD11b expression. Dibutyryl cAMP (cAMP agonist) and forskolin (cAMP-increasing agent) augmented the VD3-dependent induction of CD157 and CD11b expression while PGE1 (cAMP-decreasing agent) inhibited it, suggesting the involvement of a cAMP-dependent mechanism in VD3-induced CD157 upregulation. Co-treatment of HL-60 cells with VD3 plus TNF-alpha or ara-C produced an additive effect on CD157 upregulation. The upregulated CD157 in the VD3-differentiated HL-60 cells was able to activate CD157-dependent tyrosine kinase signal when cross-linked with anti-CD157 antibody.     

ATF3 negatively regulates adiponectin receptor 1 expression

Adiponectin is an adipocyte-derived hormone that has antidiabetic and antiatherogenic effects through two membrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Although it has been reported that the expression of AdipoR1 and AdipoR2 is regulated under physiological and pathophysiological states, their regulation is largely unknown. Previously, we demonstrated that endoplasmic reticulum (ER) stress or obesity-inducible ATF3 negatively regulates the expression of adiponectin and AdipoR2. Here, we investigated the regulation of another adiponectin receptor, AdipoR1 by ATF3, to determine if ATF3 may contribute to impairment of adiponectin signaling by repressing the expression of both adiponectin and adiponectin receptors. We found that treatment with thapsigargin, a stimulator of ATF3 expression as an inducer of ER stress, decreased AdipoR1 expression in insulin-sensitive cells (HepG2, C2C12) and insulin secreting cells (MIN6N8). Furthermore, overexpression of lentivirus carrying-ATF3 decreased AdipoR1 expression in those cells, demonstrating that ATF3 downregulates AdipoR1 expression. Next, we investigated the effects of ATF3 on human AdipoR1 promoter activity and identified an ATF3-responsive region in the promoter. Both thapsigargin treatment and ATF3 expression repressed AdipoR1 promoter activity. Transfection studies using mutant constructs containing 5′-deletions in the human AdipoR1 promoter revealed that putative ATF/CRE site is located between the −248 and −224, TGACGCGG. Chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to human AdipoR1 promoter spanning from −248 to −224. Finally, deletion of the putative ATF/CRE site abrogated ATF3-mediated transrepression of the AdipoR1 promoter. Importantly, ATF3 expression was increased in hyperglycemia or TNF-α-treated C2C12 cells in which AdipoR1 expression was decreased, suggesting that ATF3 may contribute to downregulation of AdipoR1 by hyperglycemia and TNF-α. Collectively, these results demonstrate that ATF3 negatively regulates human AdipoR1 expression via binding to an ATF3-responsive region in the promoter, which plays an important role in attenuation of adiponectin signaling and induction of insulin resistance.     

Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.     

Phosphatidylinositol-3 phosphatase myotubularin-related protein 6 negatively regulates CD4 T cells

Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14⁺ monocyte/macrophages (M/M_Ø) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/M_Ø, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/M_Ø incubated with HCV-expressing hepatocytes, as well as in primary M/M_Ø or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/M_Ø. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.     

Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

LPA signaling via LPA receptors [LPA receptor-1 (LPA₁)–LPA₆] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA₅ in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA₅ negatively regulates the cell motile and invasive activities of human sarcoma cells.     

Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8(+) T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3(+) CD8(+) T cells to make perforin and 2) the direct ability of Tim-3(+) CD8(+) T cells to kill autologous HIV infected CD4(+) target cells. Surprisingly, Tim-3(+) CD8(+) T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8(+) T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4(+) T cells and d) their ability to suppress HIV infection of CD4(+) T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8(+) T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8(+) T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.     

CD36 antisense expression in 3T3-F442A preadipocytes

An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretinsensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF3 8 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation.