Cyclic Adenosine 3',5'-Monophosphate Elevation and Biological Signaling through a Secretin Family Gs-Coupled G Protein-Coupled Receptor Are Restricted to a Single Adenylate Cyclase Isoform.

PC12 cells express five adenylate cyclase (AC) isoforms, most abundantly AC6 and AC7. These two ACs were individually silenced using lentiviral short hairpin RNAs, which lead to a decrease (≥80%) of the protein product of each transcript. These stable PC12 sublines were then used to examine potential AC isoform preference for signaling through a family B G protein-coupled receptor (GPCR). Cells were challenged with the endogenous agonist of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, or the diterpene forskolin as an AC-proximal control. Intracellular cAMP levels were elevated by forskolin about equally in wild-type, AC6, and AC7 knockdown cells. The ability of PACAP-38 and forskolin to activate three cAMP sensors downstream of AC [protein kinase A (PKA), exchange protein activated by cAMP (Epac) 2/Rapgef4, and neuritogenic cAMP sensor (NCS)/Rapgef2] was examined by monitoring the phosphorylation status of their respective targets, cAMP response element-binding protein, p38, and extracellular signal-regulated kinase. Forskolin stimulation of each downstream target of cAMP was unaffected by knockdown of either AC6 or AC7. PACAP-38 activation of all downstream targets of cAMP was unaffected by AC7 knockdown, but abolished following AC6 knockdown. Membrane cholesterol depletion with methyl-ß-cyclodextrin mimicked the effects of AC6 silencing on PACAP signaling, without attenuating forskolin signaling. These data suggest that vicinal constraint of the GPCR PAC1 and AC6 determines the exclusive requirement for this AC in PACAP signaling, but that the coupling of the cAMP sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2, to their respective downstream signaling targets, determines how cAMP signaling is parcellated to physiologic responses, such as neuritogenesis, upon GPCR-Gs activation in neuroendocrine cells.

The host range of gammaretroviruses and gammaretroviral vectors includes post-mitotic neural cells.

BACKGROUND: Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. METHODOLOGY/PRINCIPAL FINDINGS: Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease.

Bcl-xL and caspase inhibition increase the survival of rat oxytocin and vasopressin magnocellular neurons in organotypic culture.

Hypothalamic magnocellular neurons (MCNs) are highly vulnerable to axotomy-induced cell death in vivo and in vitro. In this study, we determined whether the anti-apoptotic agent Bcl-xL, a member of the Bcl-2 family which prevents programmed cell death in the central nervous system, can rescue oxytocin (OT) and vasopressin (VP) MCNs in the supraoptic nucleus (SON) in organotypic culture. We found that the novel, membrane permeant form of Bcl-xL that we employed in these studies protected both OT and VP MCNs from degeneration as long as the Bcl-xL was present in the medium. In contrast, z-VAD-fmk, an inhibitor of caspases that are involved in apoptosis, was less effective in that it significantly rescued OT MCNs (P < 0.01) but not VP MCNs (P > 0.09). Unlike the Bcl-xL, Z-VAD-fmk's effectiveness in reducing MCN cell death was not sustained for the full 15 days in vitro.

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells.

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

Antiapoptotic action of hypoxia-inducible factor-1 alpha in human endothelial cells.

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved in the adaptive response to hypoxia and consists of HIF-1 alpha and HIF-1 beta subunits. Indirect evidence suggests that HIF-1 alpha may exert both proapoptotic and antiapoptotic actions in response to hypoxia. In this study, we evaluated the effects of RNA interference (RNAi) targeting HIF-1 alpha messenger RNA (mRNA) on apoptosis in primary cultured human umbilical vascular endothelial cells (HUVECs) exposed to anoxia and reoxygenation (A/R). HUVECs were transfected with specific 21-nt small interfering RNA (siRNA) duplexes targeting HIF-1 alpha mRNA sequences or scrambled RNA duplexes and subjected either to normoxia for 251/2 h or to anoxia for 11/2 h, and subsequently normoxia for 24 h (A/R). Control samples were subjected to A/R but not transfected. HUVECs apoptosis was evaluated by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and by activated caspase-3 immunostaining and immunoblotting. The efficacy of RNAi was assessed by knockdown of HIF-1 alpha mRNA and protein expression via in situ hybridization, real-time quantitative PCR, immunohistochemistry, and Western blotting. When compared with normoxic cultures, A/R significantly upregulated HIF- 1 alpha mRNA and protein expression in HUVECs, but did not appreciably alter the percentage of apoptotic cells. In contrast, a significantly greater proportion of HUVECs transfected with specific siRNA duplexes and exposed to A/R demonstrated evidence of apoptosis when compared with nontransfected cells. Transfection with specific siRNA duplexes knocked down HIF-1 alpha mRNA and protein expression in A/R-treated cells by approximately 60%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF-1 alpha expression. These findings strongly suggest that HIF-1 alpha exerts an antiapoptotic role in HUVECs stressed by anoxia.

Extracellular Bad fused to toxin transport domains induces apoptosis.

Bad, a proapoptotic member of the Bcl-2 family, is inactivated by phosphorylation, and this loss of activity may contribute to the malignancy of certain types of tumors such as glioblastoma and prostate cancer. To determine whether extracellular Bad can be delivered into cells via cell surface receptor binding and induce apoptosis, we genetically fused the mouse Bad gene to the gene for the translocation and receptor-binding domains of diphtheria toxin (DTTR). The purified Bad (wild-type)-DTTR protein showed cytotoxicity to human glioma cells in a dose-dependent manner. Bad phosphorylation sites at codons 112 and 136 were mutated from serine to alanine to prevent Bad inactivation by kinases and to increase the toxicity of Bad. The Bad (S112A S136A)-DTTR protein was at least 5 times more toxic than Bad (wild-type)-DTTR with an IC(50) of 5 x 10(-8) M. The Bad (S112A S136A)-DTTR protein altered the subcellular distribution of Bcl-X(L), indicating that it enters the cell cytoplasm and binds Bcl-X(L). Bad (S112D S136A)-DTTR, mutated to mimic phosphorylation of Bad, showed lower toxicity than either Bad (wild-type)-DTTR or Bad (S112A S136A)-DTTR, additionally indicating that Bad-DTTR must bind Bcl-X(L) to stimulate apoptosis. We conclude that extracellular Bad can be delivered into cells via the transport domain of a bacterial toxin and may be used to induce apoptosis.