Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure.

RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation.

Analysis of the Rit subfamily GTPase-mediated signaling and neuronal differentiation and survival.

The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from the pathways that are regulated by other Ras family GTPases. Rit proteins have been demonstrated to be essential regulators in neuronal differentiation and survival.

Putting the Rit in cellular resistance: Rit, p38 MAPK and oxidative stress.

Cells mobilize diverse signaling pathways to protect against stress-mediated injury. Ras family GTPases play critical roles in this process, controlling the activation and integration of multiple regulatory cascades. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also promote cell survival.

Rit subfamily small GTPases: regulators in neuronal differentiation and survival.

Ras family small GTPases serve as binary molecular switches to regulate a broad array of cellular signaling cascades, playing essential roles in a vast range of normal physiological processes, with dysregulation of numerous Ras-superfamily G-protein-dependent regulatory cascades underlying the development of human disease. However, the physiological function for many "orphan" Ras-related GTPases remain poorly characterized, including members of the Rit subfamily GTPases. Rit is the founding member of a novel branch of the Ras subfamily, sharing close homology with the neuronally expressed Rin and Drosophila Ric GTPases.

Rit-mediated stress resistance involves a p38-mitogen- and stress-activated protein kinase 1 (MSK1)-dependent cAMP response element-binding protein (CREB) activation cascade.

The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress.

Rem-GTPase regulates cardiac myocyte L-type calcium current.

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca(2+)-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested.

Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca(2+) current (I(Ca,L)) via LTCC in murine cardiomyocytes.

Ras family small GTPase-mediated neuroprotective signaling in stroke.

Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined.

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress.

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival.

Src-dependent TrkA transactivation is required for pituitary adenylate cyclase-activating polypeptide 38-mediated Rit activation and neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Galpha/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear.

Rit signaling contributes to interferon-gamma-induced dendritic retraction via p38 mitogen-activated protein kinase activation.

The proinflammatory cytokine interferon-gamma (IFNgamma) alters neuronal connectivity via selective regressive effects on dendrites but the signaling pathways that mediate this effect are poorly understood. We recently demonstrated that signaling by Rit, a member of the Ras family of GTPases, modulates dendritic growth in primary cultures of sympathetic and hippocampal neurons. In this study, we investigated a role for Rit signaling in IFNgamma-induced dendritic retraction.

Pituitary adenylate cyclase-activating polypeptide 38-mediated Rin activation requires Src and contributes to the regulation of HSP27 signaling during neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neuropeptide that acts through G-protein-coupled receptors. While it is well established that PACAP mediates both neurotrophic and neurodevelopmental effects, the signaling cascades that underlie these diverse actions remain incompletely characterized. Here we show that the Ras-related Rin GTP-binding protein, a GTPase that is expressed predominantly in neurons, is regulated by PACAP38 signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to PACAP38-mediated pheochromocytoma cell differentiation.

Rit mutants confirm role of MEK/ERK signaling in neuronal differentiation and reveal novel Par6 interaction.

Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery.

The novel GTPase Rit differentially regulates axonal and dendritic growth.

The Rit GTPase is widely expressed in developing and adult nervous systems, and our previous data with pheochromocytoma cells implicate Rit signaling in NGF-induced neurite outgrowth. In this study, we investigated a role for Rit in neuronal morphogenesis. Expression of a dominant-negative (dn) Rit mutant in hippocampal neurons inhibited axonal growth but potentiated dendritic growth.

A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G(s)alpha protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion.

Analysis of Rit signaling and biological activity.

Rit (Ras-like expressed in many tissues) is the founding member of a novel subgroup within the larger Ras superfamily of small GTP-binding proteins. Although Rit shares more than 50% amino acid identity with Ras, it contains a unique effector domain in common with the closely related Rin and Drosophila Ric proteins and lacks the C-terminal lipidation motifs critical for the membrane association and biological activity of many Ras proteins. Interestingly, whereas Rit has only modest transforming ability when assayed in NIH 3T3 cells, Rit exhibits neuronal differentiation activities comparable to those of oncogenic mutants of Ras when assayed in PC12 and other neuronal cell lines.

Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation.

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development.

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades.

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways.

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling.

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs).

[Molecular mechanism of metastasis inhibition of nasopharyngeal carcinoma cell CNE-2L2 induced by reduction of 6A8 alpha-mannosidase expression].

Objective: To study the effect of inhibition of 6A8 alpha-mannosidase expression on adhesiveness among and E-cadherin expression on CNE-2L2 cells, and on metastasis of the tumors from the cells inoculated in nude mice.

Methods: Anchorage-independent adhesion among cells was examined in soft agar culture. E-cadherin expression was studied by immunofluorescence staining, immunohistological staining and RT-PCR.

Suppression of 6A8 alpha-mannosidase gene expression reduced the potentiality of growth and metastasis of human nasopharyngeal carcinoma.

Suppression of alpha-mannosidases by chemicals has been shown to reduce the potentiality of growth and metastasis of various tumors. In our study, the effect of 6A8 alpha-mannosidase (MAN 6A8), recently discovered in our laboratory, on malignant behaviors of tumor cells was examined. Since the suppressive effect of chemicals on alpha-mannosidase is not specific, antisense technique was used to specifically inhibit expression of the MAN 6A8 in human nasopharyngeal carcinoma cells, CNE-2L2.

[Inhibition of 6A8 alpha-manosidase expression induces decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2].

Objective: To investigate the inhibitory effect of 6A8 alpha-manosidase expression on the adhesiveness of CNE-2L2 cells to laminin and the lamellipodia on cell surface.

Methods: 6A8 alpha-manosidase expression was detected by Western blotting. For assaying the adhesion of cells to laminin, cells were incubated in laminin-coated plate at 37 degrees C for 1 h, the adhered cells were stained with crystal purple dissolved in 0.

[Inhibition of ER alpha-mannosidase expression causes reduction and shortening of microvilli on rat liver epithelial cell WB-F344].

Objective: To study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.

Methods: Recombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine.

Inhibition of 6A8 alpha-mannosidase causes oncosis-like death of BJAB cells.

To study relationships between glycosylation and cell survival we used a human alpha-mannosidase, 6A8, recently discovered in our laboratory. We found that glycosylation of BJAB cells was modified when cells were transduced with a fragment of antisense 6A8 cDNA, which resulted in an inhibition of 6A8 alpha-mannosidase expression. In comparison to wild type, mock- or sense 6A8-tansduced cells, the antisense 6A8-transduced cells showed increased cell death in culture.