John Meshki

Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

We have investigated the effect of neurokinin 1 receptor (NK1R) agonists on HEK293 cells transfected with the NK1R receptor. The NK1R receptor mediates dramatic shape changes that include contractions of the membrane cortex resulting in membrane bleb formation. We have found that the cell shape changes correlate with changes in electrical impedance measured in cellular monolayers. The shape and impedance changes were prevented after preincubation with NK1R antagonists aprepitant and L-73060. Although bleb formation usually heralds apoptotic cell death, we have found that NK1R-mediated cellular blebbing does not associate with apoptosis. Preincubation with a cell-permeable derivative of C3 transferase that blocks Rho or with the Rho-associated coiled-coil kinase inhibitor Y27632 completely prevented NK1R-induced shape and impedance changes. Blebbing was also completely inhibited by ML-9, a myosin light chain kinase inhibitor. Furthermore, the phospholipase C inhibitor U73,122 did not interfere with the effect of Substance P (SP) on cellular morphology and cellular impedance but completely blocked SP-induced intracellular calcium increase, indicating that the blebbing is a process independent of intracellular calcium elevations. Blebbing is a protein kinase C-independent process, since the nonselective protein kinase C inhibitor GF109203X did not interfere with SP-induced effects. Based on these results, we provide the first evidence that NK1R receptor-ligand interaction can cause apoptosis-independent cellular blebbing and that this process is mediated by the Rho/Rho-associated coiled-coil kinase pathway.

Hallmarks for senescence in carcinogenesis: novel signaling players.

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.

Regulation of Prostate Cancer Cell Survival by Protein Kinase Cϵ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cϵ (PKCϵ), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCδ-mediated release of death factors, the involvement of PKCϵ in this response is not known. PKCϵ depletion by RNAi or expression of a dominant negative kinase-dead PKCϵ mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFα. On the other hand, overexpression of PKCϵ by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCϵ RNAi depletion significantly enhanced the release of TNFα in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser112 in PKCϵ-depleted LNCaP cells, whereas PKCϵ overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser112 in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCϵ accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCϵ in vitro. Our results established that PKCϵ modulates survival in prostate cancer cells via multiple pathways.

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

Anti-HIV-1 activity of the neurokinin-1 receptor antagonist aprepitant and synergistic interactions with other antiretrovirals.

Objectives:Neurokinin-1 receptor (NK1R) antagonists interfere with binding of neuropeptide substance P to NK1R and exhibit novel anti-HIV-1 activities. Since NK1R antagonists effectively penetrate the blood–brain barrier to reduce the inflammatory response within the brain, we wished to evaluate their potential as anti-HIV-1 candidates for targeting HIV-1 infections of the central nervous system. Design:A series of small molecule agents were evaluated for anti-NK1R and anti-HIV-1 activity using peripheral blood mononuclear cells (PBMCs). The most promising of these, aprepitant (Emend, Merck and Co. Inc.), was investigated for potential synergies with other antiretroviral drugs. Methods:Anti-NK1R activity was tested by measuring intracellular calcium increase triggered by substance P. Anti-HIV-1 activity was evaluated by measuring p24 antigen in culture supernatants of PBMC following exposure to HIV. The concentration of drug which produced 50% reduction in intracellular calcium levels or viral production in 7-day PBMC cultures was determined. The combined effect of aprepitant with each of the major classes of anti-HIV-1 drugs was evaluated in synergy studies. Results:Aprepitant had the highest anti-HIV-1 activity of the NK1R antagonists examined and was equally active against all major HIV-1 subtypes. Aprepitant acted synergistically with protease inhibitors (ritonavir and saquinavir), but not with nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, or viral entry inhibitors. Conclusion:The ability of aprepitant to penetrate the blood–brain barrier, its safety record as an FDA-approved drug for reducing nausea and vomiting in chemotherapy, and synergistic activity with other anti-HIV-1 drugs make it a promising candidate for treatment of HIV infection.

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Substance P–mediated chemokine production promotes monocyte migration

The neuropeptide SP has physiologic and pathophysiologic roles in CNS and peripheral tissues and is involved in crosstalk between nervous and immune systems in various conditions, including HIV and SIV infection. Increased SP levels were demonstrated in plasma of HIV+ individuals as well as in the CNS of SIV‐infected, nonhuman primates. SP increases HIV infection in macrophages through interaction with its receptor, NK1R. The SP effect on immune system is both pro‐ and anti‐inflammatory and includes up‐regulation of a number of cytokines and cell receptors. The main goal of this study was to determine whether there is interplay between monocyte exposure to SP and recruitment into sites of inflammation. We now demonstrate that exposure of either human macrophages or PBMCs to SP leads to increased production of chemokines, including MCP‐1, for which expression is limited to cells of the myeloid lineage. This effect is inhibited by the NK1R antagonist, aprepitant. Exposure to conditioned medium derived from SP‐treated PBMCs resulted in increased monocyte migration through semipermeable membranes and an in vitro human BBB model. Monocyte migration was blocked by anti–MCP‐1 antibodies. Our results suggest that increased SP levels associated with HIV and other inflammatory conditions may contribute to increased monocyte migration into the CNS and other tissues through a MCP‐1–dependent mechanism.

Analog of Somatostatin Vapreotide Exhibits Biological Effects in vitro via Interaction with Neurokinin-1 Receptor

Objectives: Vapreotide, a synthetic analog of somatostatin, has analgesic activity most likely mediated through the blockade of neurokinin-1 receptor (NK1R), the substance P (SP)-preferring receptor. The ability of vapreotide to interfere with other biological effects of SP has yet to be investigated. Methods: We studied the ability of vapreotide to antagonize NK1R in three different cell types: immortalized U373MG human astrocytoma cells, human monocyte-derived macrophages (MDM) and a human embryonic kidney cell line, HEK293. Both U373MG and MDM express endogenous NK1R while HEK293 cells, which normally do not express NK1R, are stably transformed to express human NK1R (HEK293-NK1R). Results: Vapreotide attenuates SP-triggered intracellular calcium increases and nuclear factor-κB activation in a dose-dependent manner. Vapreotide also inhibits SP-induced interleukin-8 and monocyte chemotactic protein-1 production in HEK293-NK1R and U373MG cell lines. Vapreotide inhibits HIV-1 infection of human MDM in vitro, an effect that is reversible by SP pretreatment. Conclusions: Our findings indicate that vapreotide has NK1R antagonist activity and may have a potential application as a therapeutic intervention in HIV-1 infection.

Substance P enhances HIV-1 infection in human fetal brain cell cultures expressing full-length neurokinin-1 receptor

The associations between the neurokinin-1 receptor (NK-1R), substance P (SP), and HIV-1 were investigated in neurosphere-derived cultures of microglial-depleted human fetal brain cells (HFBC). Full-length NK-1R was identified in HFBC cultures. SP treatment of the HFBC increased intracellular calcium mobilization and decreased electrical impedance, both of which were blocked by the NK-1R antagonist aprepitant. SP treatment of HIV-1-infected HFBC upregulated HIV-1 expression. These data show that human neural cells grown from neurospheres express functional full length NK-1R that is responsive to SP, and that SP enhanced HIV-1 infection in HBFC.