Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation

Abstract

The development of methods for engineering proteins with novel properties opens the way to manipulating intracellular processes in a therapeutically useful way. Glucocorticoids, acting via glucocorticoid receptors (GR), are potent anti-inflammatory agents, acting to oppose nuclear factor kappa B (NF B) function. The herpes viral protein, VP22, has been reported to confer intercellular trafficking activity on 'cargo' proteins, potentially facilitating gene therapy with intracellular proteins.VP22GR, resulting from the addition of VP22 to the N terminal of GR, was equipotent with the wild-type GR in opposing NF B p65-driven expression of an NF B reporter gene. Surprisingly, VP22GR was incapable of inducing transactivation of positive glucocorticoid reporter genes (MMTV-luc and TAT3-luc). Furthermore, the VP22GR had powerful dominant negative activity on both endogenous and exogenous GR transactivation. VP22GR was cytoplasmic in quiescent cells, and after hormone addition underwent nuclear translocation to share the same distribution as the GR. The ability of the VP22GR to selectively confer and enhance glucocorticoid-dependent transrepression of NF B may be of use therapeutically in e.g. transplant rejection, inflammatory arthritis or asthma.

Glucocorticoids suppress macrophage migration inhibitory factor (MIF) expression in a cell-type-specific manner

“…This also accords with our earlier observations in the two cell lines, which showed that CEM C7A cells required higher concentrations of Dex to maximally activate a positive GC reporter gene (Soden et al 2002). We have previously reported that inhibition of NF B actions by GC occurs at lower concentrations of ligand than gene transactivation (Ray et al 1999).…”

“…Both cell lines express the GR, and we have previously shown that both cell lines are sensitive to GC by transfection of reporter genes (Soden et al 2002). GC does not affect A549 cell death (Webster et al 2002).…”

“…SV40-luc, containing the SV40 promoter is the same pGL3-luc plasmid, was purchased from Promega. The glucocorticoid response MMTV-luc reporter gene was previously described (Soden et al 2002, Stevens et al 2003.…”

Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line

“…This is a simple, glucocorticoid-activated gene that has been extensively characterised (Iniguez-Lluhi et al 1997, Soden et al 2002. The AF-1 and AF-2 domains of GR act differently at the simple TAT3 reporter in COS 7 cells, in that deletion of AF-2 allows constitutive transactivation, while removal of AF-1 produces a C-terminal GR construct which shows ligand-dependent transactivation (Soden et al 2002). In COR L103 cells, deletion of the AF-2 domain of the GR caused ligand-independent transactivation, as expected, but with a small induction in the presence of dexamethasone.…”

Poor intercellular transport and absence of enhanced antiproliferative activity after non‐viral gene transfer of VP22‐P53 or P53‐VP22 fusions into p53 null cell lines in vitro or in vivo

“…Several studies have already reported altered functions with fusion at the Cterminal extremity of VP22 [7,33,34]. For instance, a VP22-CD fusion was unable to spread whereas a CD-VP22 fusion showed efficient intercellular transport [7] and a VP22-GR (glucocorticoid receptor) protein was incapable of inducing transactivation of positive reporter genes and even demonstrated powerful dominant-negative activity on both endogenous and exogenous GR transactivation [33].…”