Functional microRNA screening using a comprehensive lentiviral human microRNA expression library.

BACKGROUND: MicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs. RESULTS: To systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs. CONCLUSIONS: With the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion.

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.

Active Rap1, a small GTPase that induces malignant transformation of hematopoietic progenitors, localizes in the nucleus and regulates protein expression.

Rap1, a member of the Ras superfamily, regulates cytoskeletal changes in lower eukaryots and integrin-mediated adhesion in hematopoietic cells. Sustained activation of Rap1 in mouse hematopoietic stem cells causes expansion of hematopoietic progenitors, followed by a myeloproliferative disorder mimicking chronic myeloid leukemia. Moreover, these mice develop a B-cell lymphoproliferative disorder resembling chronic lymphocytic leukemia. Here, we used HEK 293 cells as a tool to examine the molecular effects of Rap1. We observed that a constitutively active Rap1 mutant localized predominantly in the nucleus. Nuclear localization of endogenous Rap1-GTP was also detected upon physiologic activation. A potential consequence of nuclear localization of Rap1-GTP is the regulation of gene expression. We used a high throughput proteomic approach to identify gene products potentially modulated by Rap1-GTP. Out of 1000 proteins examined, 64 proteins were upregulated and 66 proteins were downregulated. The differentially expressed gene products belong to cytoskeletal regulator proteins, signaling molecules, transcription factors, viability regulators, and protein transporters. This analysis provides the first fingerprint of gene product expression regulated by Rap1 and may contribute to our understanding of malignant transformation mechanisms regulated by this small GTPase.

Induction of immunologic tolerance for allogeneic hematopoietic cell transplantation.

The ability to achieve complete hematopoietic engraftment in the allogeneic setting without intensive myeloablative chemotherapy will have a profound effect on the practice of allogeneic hematopoietic cell transplantation (HCT). Novel methods to induce antigen-specific T-cell tolerance provide promise to ensure engraftment and reduce GVHD without producing generalized and other toxicities caused by myeloablative conditioning regimens. Compelling experimental evidence indicates that the antigen receptors on T-lymphocytes have dual potential to transmit crucial activation signals for initiating immune responses and to discharge equally potent inactivating signals to abort or inhibit immune responses. Many events impact on this fundamental decision-making process and one of the great challenges for modern immunology is to decipher the molecular wiring that integrates and converts the extrinsic and intrinsic variables into positive or negative cellular responses termed immunity and anergy, respectively. Our currently expanding understanding of the biochemical and molecular basis of T-cell anergy provides great promise to improve our ability to design novel clinical therapeutic approaches in order to induce antigen-specific tolerance in vivo. Importantly, strategies now exist to segregate graft versus tumor (GVT) effects from GVHD. Therefore, achievement of limited and specific tolerance to host alloantigens by selectively inactivating the indicated subsets of alloantigen-specific T-lymphocytes will prevent GVHD but retain the GVT effect of the graft. Such treatment approaches will expand the donor pool, because they will allow transplantation between individuals with increasing human leukocyte antigen (HLA) disparity, enable reduction of the need for non-specific immunosuppression, and reduce the risk of opportunistic infections and relapse of leukemia.

Correction: A Functional Screen Identifies Specific MicroRNAs Capable of Inhibiting Human Melanoma Cell Viability.

[This corrects the article DOI: 10.1371/journal.pone.0043569.].

A functional screen identifies specific microRNAs capable of inhibiting human melanoma cell viability.

Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

cAMP inhibits both Ras and Rap1 activation in primary human T lymphocytes, but only Ras inhibition correlates with blockade of cell cycle progression.

Cyclic adenosine monophosphate (cAMP) is a negative regulator of T-cell activation. However, the effects of cAMP on signaling pathways that regulate cytokine production and cell cycle progression remain unclear. Here, using primary human T lymphocytes in which endogenous cAMP was increased by the use of forskolin and 3-isobutyl-1-methylxanthine (IBMX), we show that increase of cAMP resulted in inhibition of T-cell receptor (TCR)/CD3 plus CD28-mediated T-cell activation and cytokine production and blockade of cell cycle progression at the G(1) phase. Increase of cAMP inhibited Ras activation and phosphorylation of mitogen-induced extracellular kinase (MEK) downstream targets extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol-3-kinase (PI3K) downstream target protein kinase B (PKB; c-Akt). These functional and biochemical events were secondary to the impaired activation of ZAP-70 and phosphorylation of LAT and did not occur when cells were stimulated with phorbol ester, which bypasses the TCR proximal signaling events and activates Ras. Increase of cAMP also inhibited activation of Rap1 mediated by TCR/CD3 plus CD28. Importantly, inhibition of Rap1 activation by cAMP was also observed when cells were stimulated with phorbol ester, although under these conditions Ras was activated and cells progressed into the cell cycle. Thus, TCR plus CD28-mediated activation of ERK1/2 and PKB, cytokine production, and cell cycle progression, all of which are inhibited by cAMP, require activation of Ras but not Rap1. These results indicate that signals that regulate cAMP levels after encounter of T cells by antigen will likely determine the functional fate toward clonal expansion or repression of primary T-cell responses.

CD28 costimulation mediates down-regulation of p27kip1 and cell cycle progression by activation of the PI3K/PKB signaling pathway in primary human T cells.

CD28 provides a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell activation, cytokine production, and clonal expansion. We have recently shown that CD28 directly regulates progression of T lymphocytes through the cell cycle. Although a number of signaling pathways have been linked to the TCR/CD3 and to CD28, it is not known how these two receptors cooperate to induce cell cycle progression. Here, using cell-permeable pharmacologic inhibitors of phosphatidylinositol 3-hydroxykinase (PI3K) and mitogen-activated protein kinase kinase (MEK1/2), we show that cell cycle progression of primary T lymphocytes requires simultaneous activation of PI3K- and MEK1/2-dependent pathways. Decreased abundance of cyclin-dependent kinase inhibitor p27(kip1), which requires simultaneous TCR/CD3 and CD28 ligation, was dependent upon both MEK and PI3K activity. Ligation of TCR/CD3, but not CD28 alone, resulted in activation of MEK targets extracellular signal-related kinase 1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target protein kinase B (PKB; c-Akt). CD28 ligation alone was also sufficient to mediate inactivating phosphorylation of PKB target glycogen synthase kinase-3 (GSK-3). Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27(kip1), hyperphosphorylation of retinoblastoma tumor suppressor gene product, and cellular proliferation. Thus, inactivation of the PI3K-PKB target GSK-3 could substitute for CD28 but not for CD3 signals. These results show that the PI3K-PKB pathway links CD28 to cell cycle progression and suggest that p27(kip1) integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 in primary T lymphocytes.

Differential localization and function of ADP-ribosylation factor-6 in anergic human T cells: a potential marker for their identification.

Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. Recent data indicate that anergic cells have a distinct gene expression program that determines their unique function. In this study we show that anergic human T cells selectively express the small GTPase ADP-ribosylation factor-6 (ARF6), which is involved in membrane traffic and regulation of the cortical actin cytoskeleton. ARF6 was expressed in the GTP-bound form that localizes at the plasma membrane, resulting in a distinct morphologic appearance of anergic cells. Forced expression of ARF6-GTP in Jurkat T cells prevented TCR-mediated reorganization of cortical actin, extracellular signal-regulated kinase1/2 activation, and IL-2 transcription. Forced expression of ARF6-GTP in primary human T cells inhibited extracellular signal-regulated kinase1/2 activation and proliferative responses. Importantly, T cells with the distribution pattern of ARF6-GTP were detected in peripheral blood, suggesting that anergic T cells may constitutively exist in vivo.