The G-protein-coupled receptor kinase 5 inhibits NFκB transcriptional activity by inducing nuclear accumulation of IκBα

Abstract

G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5–IκBα interaction on NFκB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IκBα, leading to the inhibition of NFκB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IκBα, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IκBα as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFκB, we evaluated the effects of GRK5-RH on NFκB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFκB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFκB activity.

G-protein-coupled receptor (GPCR) kinases, GRKs, constitute a large family of serine/threonine protein kinases that regulate GPCR signaling (1–3), consisting of 7 isoforms that share structural and functional similarities (4). A central catalytic domain is flanked by an N-terminal domain that includes a region of homology to regulators of G-protein signaling (RH) and a C-terminal domain of variable length (5, 6). The catalytic domain of GRKs is relatively well-conserved among the members of different subfamilies (≈45% sequence identity), whereas N-terminal RH domains display weak homology (≈27%) and C termini have little or no sequence homology. GRKs have different tissue distribution, subcellular localization, and kinase activity regulation (7, 8). GRKs mostly localize at the plasma membrane (2), but recently Johnson et al. (7) demonstrated that GRK4–6 (but not other GRKs) can shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence (NES), thus suggesting a nuclear effect for the GRK4–6 subfamily.

NFκB is an ubiquitously expressed and highly regulated dimeric transcription factor (3) regulating the expression of genes responsible for innate and adaptive immunity, tissue regeneration, stress responses, apoptosis, cell proliferation, and differentiation (9–14). Within the canonical view of the regulation of the NFκB activity, the inactive NFκB/IκBα complex localizes in the cytosol until an extracellular stimulus, such as TNFα or LPS, induces IκB kinase (IKK)-mediated IκBα phosphorylation and subsequent degradation by the proteasome (15). NFκB is therefore free to shuttle into the nucleus and to bind to specific sequences in the promoter or enhancer regions of target genes (11). Recently this view has been partially revisited by the description of NES and NLS sequences on the NFκB/IκBα complex that allow a continuous shuttling between the nucleus and the cytosol by means of nuclear transporters, such as CRM1 (1, 3). New partners of this complex are described along the way: β-arrestin 2 interacts with the NFκB/IκBα complex within the cytosol, causing the blockade of NFκB transcriptional activity (16, 17). Studying a NFκB cognate precursor—NFκB p105, which actively participates in cytosolic signal transduction but lacks transcriptional activity—Parameswaran et al. (18) recently described the ability of GRK5 to physically interact with NFκB p105. These authors did not investigate the eventual effects of GRK5 on NFκB transcriptional activity.

NFκB drives VEGF, bFGF, IL-8, and other cytokines' expressions (19). In the endothelium, cytokine production sustains tissue regeneration by providing the start to neoangiogenesis and therefore blood and nutrient support. Failure of NFκB activation is associated with impaired cytokine production and angiogenesis (20, 21).

On the basis of these preexisting observations, we hypothesized that GRK5 could regulate NFκB transcriptional activity. In particular, we investigated whether GRK5 affects IκBα cellular levels and NFκB activity and the molecular basis for GRK5 and IκBα physical interaction. Also, we assessed the effects on NFκB-dependent phenotypes such as apoptosis, cytokine production, and in vivo and in vitro angiogenesis.

Results

GRK5 Overexpression Causes IκBα Nuclear Accumulation.

We evaluated GRK5 and IκBα cellular localization by Western blot in bovine aorta endothelial cells (BAEC) overexpressing the WT bovine GRK5 gene. The overexpression of GRK5-WT increases IκBα levels in whole-cell extracts by inducing IκBα nuclear accumulation and does not change cytosol levels (Fig. 1A). Fig. 1B shows that the total and nuclear levels of GRK5 after transfection increase in a time-dependent manner and are associated with a progressive accumulation of IκBα levels both in whole-cell lysates and nuclear extracts. Accordingly, NFκB activity decreases over time, synchronized with the increase of IκBα levels. These results suggest that small increments of GRK5 are enough to induce IκBα nuclear accumulation and NFκB activity inhibition. Indeed, transfection of as low as 0.5 μg of GRK5 induces an increase of IκBα levels in the cell (Fig. 1C). GRK5-induced IκBα nuclear accumulation occurs also in HEK293 cells, where GRK5-WT overexpression increases IκBα levels and inhibits NFκB activity. In these cells, for which there is available a GRK5 siRNA (22), GRK5 knockdown leads to the reciprocal effect, which is the reduction of the cellular levels of IκBα and the increase of NFκB activity (Fig. 1D). Increasing doses of GRK5 siRNA is associated with a progressive reduction of IκBα and inhibition of NFκB activity (Fig. 1E).

Fig. 1.

GRK5 binds and stabilizes IκBα, causing inhibition of NFκB activity. (A) IκBα and GRK5 levels were analyzed in whole, cytosolic, and nuclear extracts by Western blot in BAEC overexpressing GRK5-WT. The overexpression of GRK5-WT increases IκBα levels in whole-cell extracts and induces IκBα nuclear accumulation. (B) To evaluate the effects of GRK5 on IκBα turnover, we analyzed GRK5 and IκBα expression in a time-course of GRK5 transfection. The increase of GRK5 levels induces a progressive increase of IκBα levels and a reduction of NFκB activity (*P < 0.05 vs. control). (C) A time course of transfection of GRK5 was performed in BAEC and GRK5 and IκBα levels analyzed both in whole- and nuclear-cell extracts. The time-dependent increase of GRK5 levels associates to IκBα stabilization and nuclear localization and subsequently NFκB activity inhibition (*P < 0.05 vs. control). (D) HEK293 transfected with GRK5-WT or GRK5siRNA were analyzed by WB for GRK5 and IκBα expression. As in BAEC, GRK5 overexpression induces IκBα accumulation; conversely, GRK5 knockdown by GRK5siRNA associates to IκBα degradation. Actin is shown for protein-loading control. HEK293 were transfected with κB-Luc plasmid and GRK5-WT or GRK5-siRNA and luciferase activity was measured. GRK5-WT overexpression causes inhibition of NFκB activity, whereas GRK5 knockdown increases NFκB transcription levels. The data in the bar graph are expressed as mean±SEM and are representative of 3 experiments (*P < 0.05 vs. control). (E) GRK5 and IκBα expression and luciferase activity were assessed in HEK293 transfected with increasing doses of GRK5siRNA. The progressive knockdown of GRK5 associates to a similar reduction in IκBα levels and NFκB activity. Actin was used as loading control (*P < 0.05 vs. control). (F) BAEC were transiently transfected with either GRK5-WT or the kinase-dead mutant GRK5-K215R. Both variants are found in the nucleus together with increased amount of IκBα and NFκB. Equal amounts of proteins are confirmed by WB for histone 3. BAEC were transfected with κB-Luc plasmid and GRK5-WT, GRK5-K215R, and GRK2, and luciferase activity was measured. Overexpression of both GRK5-WT or GRK5-K215R significantly inhibit NFκB transcription activity in BAEC, whereas GRK2 overexpression has no inhibitory effect. The data are expressed as mean±SEM and are representative of three experiments (*P < 0.05 vs. control).

To evaluate whether the catalytic activity of GRK5 is needed for IκBα nuclear accumulation, we overexpressed in BAEC the kinase-dead mutant of GRK5 (K215R). GRK5-WT and GRK5-K215R are both found in appreciable amounts in the nucleus (Fig. 1F). Both proteins cause a similar increase of IκBα and NFκB levels in the nucleus and NFκB activity inhibition, whereas the overexpression of the WT human GRK2 does not inhibit NFκB activity (Fig. 1F). This evidence suggests that the catalytic activity of GRK5 is not required to produce IκBα-mediated NFκB inhibition, as demonstrated by the lack of changes of IκBα phosphorylation after the overexpression of GRK5 (Fig. S1). This latter finding also rules out the role of other kinases such as IKK. Because GRK5 is a well-known regulator of GPCR signaling, we explored the possibility of a correlation between IκBα regulation and GPCR activation by evaluating GRK5 and IκBα levels after agonist-induced activation of β-adrenergic receptors (βARs) with isoproterenol (ISO, 10⁻⁷M). Interestingly, ISO induces GRK5 and IκBα nuclear translocation (Fig. S2), suggesting that βARs regulate NFκB activity through GRK5.

GRK5 Interacts with IκBα.

Because the kinase activity appears to be not necessary to GRK5-induced IκBα nuclear accumulation, we explored the possibility of a direct interaction between GRK5 and IκBα. In BAEC, GRK5 is detected in immunoprecipitates of both IκBα and NFκB, suggesting that GRK5 does indeed bind IκBα/NFκB complex (Fig. S3A).

In resting cells, IκBα is linked to NFκB; to rule out the possibility that the IκBα-GRK5 interaction is mediated by NFκB, we explored the ability of GRK5 to bind IκBα directly. To this aim, we used purified GRK5, GRK2, MEK-I, and IκBα proteins for an in vitro binding assay; MEK-I was chosen among the available purified kinases because it is structurally distant from GRKs. As for GRK2, we analyzed its ability to interact with IκBα, because both GRK2 and GRK5 are considered prototypes of different GRK subfamilies for important structural dissimilarities in the C terminus. Both purified GRK2 and GRK5 coimmunoprecipitate with IκBα (Fig. S3B), whereas MEK-I does not, indicating specific affinity between GRKs and IκBα. The direct interaction was confirmed in an overlay assay with purified proteins by using IκBα as bait (Fig. S3C), showing that GRK5 binds IκBα with a 5-fold larger affinity than GRK2 (GRK2: 0.89 ± 0.09; GRK5: 4.78 ± 0.7 densitometric units; P < 0.05, t Test). Therefore, the region of interaction between GRKs and IκBα lies either in the N-terminal region that comprises the RH domain or in the catalytic domain, because they both are partially maintained between GRK2 and GRK5.

RH Is the Interacting Domain of GRK5 with IκBα.

To map the IκBα binding region on GRK5 we generated myc/histidine-tagged, truncated mutants of GRK5, including GRK5-WT amino acids 1–590, GRK5-NT amino acids 1–176, GRK5-RH amino acids 50–176, and GRK5-CT amino acids 176–590 (Fig. 2A and Fig. S4). In BAEC, GRK5-WT, -NT and -RH (but not -CT) are all able to coprecipitate IκBα (Fig. 2B), indicating the RH as the IκBα-interacting domain of GRK5. To further explore this point, we overexpressed in BAEC the GRK5-RH domain and then verified the ability of IκBα to coimmunoprecipitate with GRK5. The overexpression of GRK5-RH competes with endogenous GRK5 in the binding to IκBα and therefore decreases the coimmunoprecipitation of IκBα and GRK5 (Fig. 2C). Also, by Western blot, we examined whether the overexpression of GRK5 domains affects IκBα levels. GRK5-NT and -RH (but not -CT) significantly increase the amount of IκBα in whole-cell (Fig. S5) and nuclear (Fig. S6) extracts. By immunofluorescence in BAEC, we observed that GRK5-WT localizes both at perinuclear and nuclear levels (Fig. S7); this compartmentalization is accompanied by a similar subcellular distribution of IκBα. GRK5-NT and -RH colocalized with IκBα in the nucleus. GRK5-CT shows a mainly cytosolic localization (Fig. S7) and so does IκBα in the same cells. We then cloned a myc/histidine-tagged, truncated mutant of IκBα (IκBα-CT) (Fig. 2A) lacking the N- terminal NES sequence and evaluated its ability to immunoprecipitate GRK5. Fig. 2D shows that IκBα-WT coimmunoprecipitates with GRK5 whereas IκBα-CT loses such ability. Both IκBα-WT and -CT preserve the ability to interact with NFκB. These data confirm that GRK5 interacts with the complex NFκB/IκBα by directly binding to the N-terminal domain of IκBα.

Fig. 2.

The RH domain of GRK5 interacts with IκBα and causes inhibition of NFκB activity. (A) To map the IκBα binding region on GRK5, we created myc/histidine-tagged, truncated mutants of GRK5 including GRK5-WT amino acids 1–590, GRK5-NT amino acids 1–176, GRK5-RH amino acids 50–176, and GRK5-CT amino acids 176–590. To verify the hypothesis that GRK5 masks the N-NES on IκBα, we created myc/histidine-tagged mutants of IκBα including IκBα-WT amino acids 1–317 and IκBα-CT amino acids 59–317. (B) In lysates from BAEC overexpressing histidine-tagged GRK5-WT, -NT, -RH, and -CT, histidine was precipitated by using Ni Sepharose beads and subjected to a Western blot with anti-IκBα or anti-His antibodies. GRK5-WT, -NT, and -RH (but not -CT) coprecipitate with IκBα. (C) Whole extracts from control and GRK5-RH transfected cells were immunoprecipitated with anti-IκBα antibody, and Western blot was performed with anti-GRK5 antibody. GRK5-RH overexpression attenuates IκBα immunoprecipitation of GRK5 (*P < 0.05 vs. control). (D) The immunoprecipitation study with IκBα mutants shows that IκBα-WT coimmunoprecipitates with GRK5 whereas IκBα-CT loses such ability. Both IκBα-WT and its mutant preserve the ability to interact with NFκB. (E) We evaluated the effects of GRK5 on NFκB activity by luciferase assay in BAEC overexpressing truncated GRK5 mutants and stimulated with LPS (1 μg/ml) for 4 h. GRK5-WT, -NT, and -RH inhibit LPS-induced NFκB transcriptional activity whereas GRK5-CT doesn't change this response (*P < 0.05). (F) To further confirm such an effect, we evaluated LPS-induced NFκB binding to DNA by electrophoretic mobility shift assay in nuclear extracts. GRK5-WT, -NT, and -RH (but not -CT) overexpression cause a significant inhibition of LPS-induced NFκB DNA binding. Data are expressed as mean±SEM (*P < 0.05 vs. control).

GRK5-RH Negatively Regulates NFκB Activity.

To evaluate the effect of GRK5 single domains on the regulation of LPS-induced NFκB activity, we performed a luciferase assay with the above-described reporter plasmid. The overexpression of GRK5-WT, -NT, and -RH (but not -CT) significantly inhibits NFκB transcription of the reporter gene both in resting cells and after LPS stimulation (1 μg/ml, 4 h) (Fig. 2E). To further confirm such effect, we evaluated NFκB transcription activity by EMSA. GRK5-WT, -NT, and -RH (but not -CT) overexpression cause a significant inhibition of LPS-induced NFκB DNA binding (Fig. 2F).

GRK5-RH Inhibits TNFα Transcription.

The biological consequences of this mechanism of regulation of NFκB activity were therefore investigated. It is well-known that NFκB regulates the expression of many cytokines, including TNFα (13, 21, 23). Because we demonstrated that GRK5-RH inhibits NFκB activation, we explored the ability of GRK5-RH to regulate cytokine expression, TNFα in particular, by means of Northern blot. GRK5-WT, -NT, and -RH, (but not -CT) inhibit TNFα mRNA expression induced by LPS stimulation (Fig. 3A).

Fig. 3.

GRK5-RH regulates different phenotypes that are under the transcriptional control of NFκB. (A) NFκB-dependent TNFα expression was assessed by Northern blot in BAEC stimulated with LPS for 4 h. GRK5-WT, -NT, and -RH (but not -CT) inhibit TNFα mRNA expression induced by LPS (*P < 0.05). (B) To evaluate the effect of GRK5-RH on apoptosis, we analyzed the cleavage of caspase 3 by Western blot. The overexpression of GRK5-RH increases cleaved caspase 3 levels, suggesting that NFκB inhibition induced by GRK5-RH causes an increase of apoptotic responses (*P < 0.05 vs. control (C) GRK5-RH overexpression causes apoptosis as shown by Annexin-V staining respective to live cells; propidium iodide and DAPI were used as controls. (D) Migration of confluent BAEC was measured after the cell monolayer was partially wiped away. The area of the migrating cells was measured in several fields of view and is shown in the graph. Data are presented as FBS-induced percentages of migration respective to resting cells (*P < 0.05 vs. FBS 5%). (E) Representative-phase contrast photomicrographs of control and GRK5-RH transiently transfected BAEC plated on Matrigel are shown. Microscopy revealed numbers of network projections formed in each group after 12 h of incubation (*P < 0.05 vs. control). Data from three experiments in triplicate are summarized in the graph.

GRK5-RH Increases Apoptosis.

Biological implications can also be observed in other phenotypes. Indeed, endothelial NFκB has been implicated in angiogenesis through its protective action of endothelial cells from apoptosis (24). NFκB is a critical regulator of apoptotic responses in different physiological and pathological contexts (25–27). The overexpression of GRK5-RH increases cleaved caspase 3 levels, a marker of activated apoptosis (Fig. 3B). Apoptosis was also assessed by fluorescence with fluorescein-conjugated Annexin-V: Cells overexpressing GRK5-RH have a larger fluorescent staining compared with control cells (Fig. 3C). Both data are therefore consistent with the concept that GRK5-RH-induced NFκB inhibition causes an increase of apoptotic responses.

GRK5-RH Inhibits Endothelial Cell Migration and Vascular Tube Formation.

We determined other proangiogenic phenotypes of BAEC that are affected by NFκB. In particular, we evaluated the effects of GRK5-RH on BAEC migration by using a cell monolayer wounding assay. In the presence of 5% FBS, BAEC display a greater capacity over unstimulated cells to migrate into the wounded area; GRK5-RH transfection inhibits the migration of FBS-induced BAEC (Fig. 3D and Fig. S8). NFκB promotes endothelial tube formation on extracellular matrix, which corresponds in vitro to capillary-network formation in vivo (28). GRK5-RH affects this ability as well by inhibiting vascular network formation on Matrigel matrix (Fig. 3E). Our in vitro results sustain the idea that GRK5-RH negatively regulates the proangiogenic responses of BAEC.

AdGRK5-NT Inhibits Regenerative Responses in Vivo.

To extend these observations to the in vivo setup, regenerative responses were obtained in rats by chronic ischemia and wound healing. Chronic ischemia in the rat is induced by femoral artery removal. We combined this technique with vascular gene transfer achieved by means of intrafemoral artery infusion of adenoviruses encoding for GRK5-NT, AdGRK5-NT (a kind gift from Walter Koch, Thomas Jefferson University, Philadelphia, PA), or a control (AdEmpty). Acute ischemia causes an inflammatory response which causes the formation of new vessels and restoration of blood supply to the ischemic zone. By live digital angiography and thrombosis in myocardial infarction (TIMI) score (29), blood flow results partially restored after 14 days in the AdEmpty ischemic hindlimb (Fig. 4A). On the contrary, in AdGRK5-NT animals this adaptative response is greatly impaired with a longer time to ischemic hindlimb perfusion (Fig. 4A). Validation of this result comes from dyed beads recovery from the rat limb muscles, after intraaortic infusion of dyed agarose beads (Fig. 4B). This impaired response is paralleled by lower NFκB-dependent TNFα expression in the ischemic muscle of the AdGRK5-NT treated hindlimb (Fig. 4C). Also, in vivo NFκB activation sustains wound healing by chemokine production such as MCP-1 and monocyte and neutrophil infiltration (30). We treated blade-imposed skin wounds with AdGRK5-NT and assessed GRK5-NT localization by immunohistochemistry with the specific antibody anti-GFP (Fig. 4D). We observed a longer time for wound-healing compared with AdEmpty (Fig. 4D). This impaired response associates with a reduction of monocyte/neutrophil infiltrate (Fig. 4E) and lower expression of the chemokine MCP-1 (Fig. 4F).

Fig. 4.

AdGRK5-NT inhibits regenerative responses in vivo. (A) After surgical removal of the femoral artery in rats, blood flow through the ischemic hindlimb is granted by neoangiogenic and vascular regenerative phenomena, mediated in part by NFκB-dependent cytokines. After 14 days of chronic ischemia, the time to perfusion of the ischemic hindlimb was assessed by digital angiographies, showing an increased TIMI score to perfusion in AdGRK5-NT respective to AdEmpty rats (*P < 0.05). (B) Blood flow was also assessed by muscle content of dyed beads after intra-arterial injection in the bloodstream. AdGRK5-NT reduces blood perfusion in ischemic hindlimb respective to AdEmpty. The ischemic-to-nonischemic ratio of dyed beads content per mg of hindlimb muscle tissue (*P < 0.05 vs. AdEmpty) is shown. (C) RNA from the hindlimbs of AdEmpty and AdGRK5-NT-treated rats was extracted by means of trizol reagent and TNFα expression was evaluated by Northern blot. The analysis shows an attenuation of cytokine expression in GRK5-NT-treated ischemic compared to AdEmpty ischemic hindlimb (*P < 0.05). (D) In another model of regeneration, the skin-wound healing, we observed a longer period for healing of wounds that are treated with AdGRK5-NT rather than AdEmpty at the time of surgery. The expression of the transgene was assessed by taking advantage of the bicistronic nature of the adenovirus, encoding also for the GFP under the CMV promoter, with the specific antibody anti-GFP. The transgene localizes both in epidermis and dermis. (E) In AdGRK5-NT-treated wounds, haematoxylin/eosin staining reveals a smaller amount of infiltrate when compared with AdEmpty control. (F) Similarly, MCP-1 expression assessed in the wound is reduced by AdGRK5-NT when compared with AdEmpty wounds.

Discussion

To date, the specific cellular function that is associated with GRKs is GPCR phosphorylation and desensitization; recent findings unveil that GRKs are able to interact with nonreceptor proteins (PI3K, AKT, and MEK), suggesting new cellular functions for these kinases (16, 31–33).

GRK5 is the paradigm of a subfamily of GRKs that possess the ability to localize within the nucleus (7). This feature suggests that this kinase may participate in nuclear functions, such as DNA duplication or transcription.

The present study identifies and characterizes an interaction of GRK5 and IκBα in endothelial cells, leading to IκBα nuclear accumulation and inhibition of NFκB transcriptional activity.

The possibility that members of the GRKs and NFκB family can physically interact has been previously proposed (www.signaling-gateway.org/data/Y2H/cgi-bin/y2h.cgi and ref. 18). None of these reports, however, has investigated the interaction of GRK5 and IκBα or the effects on nuclear transcriptional activity of NFκB. Therefore, our results greatly expand the previously reported data by identifying the role of GRK5 on NFκB activity and pointing to IκBα as the partner of GRK5 in the regulation of NFκB signaling. We also demonstrated that both GRK5 and GRK2 interact with IκBα but that GRK2 interaction is weaker than GRK5 and has no effects on NFκB activity. The weak homology of GRKs in the NT domain, in presence of elevated amounts of protein, probably makes GRK2 able to perform the same protein–protein interaction of GRK5. This homology can also explain the interaction between GRK2 and NFκB shown by the Alliance for Cellular Signaling.

Using truncated GRK5 mutants, we identify RH as the GRK5 domain involved in IκBα binding, nuclear accumulation, and inhibition of NFκB activity and DNA binding.

Our results, together with previous results, point to a protein–protein interaction as the regulatory mechanism for NFκB activity, similar to the effect of β-arrestin 2 on NFκB. Indeed, β-arrestin 2 inhibits NFκB activity (15, 34, 35) by stabilizing IκBα levels (34). The prevalent localization of β-arrestin 2 in the cytosol explains the finding of Gao et al. (16) that β-arrestin 2 overexpression decreases NFκB nuclear translocation and blocks the IκBα/NFκB complex in the cytosol. Our results differ from this previous report because we demonstrated that GRK5 accumulates in the nucleus, where it stabilizes the IκBα/NFκB complex. These results suggest that GRK5 and β-arrestin 2 regulate IκBα turnover by different mechanisms in different subcellular compartments. Although the canonical interpretation of NFκB/IκBα regulation depicts the interaction between these 2 molecules in the cytosol, a dynamic model has been recently proposed explaining that the IκBα/NFκB complex shuttles in and out of the nucleus, although it mainly stays in the cytosol due to active export by the nuclear transporter CRM-1 (36), which recognizes the NES at the C-terminal domain of IκBα. Our results fit this model because the GRK5-RH binds to IκBα in its N-terminal region and probably masks the NES on IκBα-CT and induces nuclear accumulation of the complex. The physiological trigger to the increase of GRK5 in the nucleus could be the activation of GPCR. Indeed, small nuclear increases of GRK5, such as those induced by ISO stimulation, are already enough to cause IκBα nuclear accumulation and NFκB inhibition. Also, other agonists can induce acute upregulation of GRK5, such as LPS in peripheral blood cells (37). The regulation of NFκB activity is pivotal to the expression of cellular genes involved in inflammation responses (13, 21, 23) and in various diseases, such as cancer (19, 23, 38–42), cardiovascular disease (39), diabetes (43, 44), and chronic inflammation (19, 42). For these reasons, we explored the eventual effects of our in vitro results in more complex models. In rats, NFκB inhibition results in the impairment of regenerative responses. Indeed, the adenoviral-mediated gene transfer of GRK5-NT to the ischemic hindlimb causes an impairment of the expected regenerative response and a decrease of NFκB-dependent transcription of TNFα. In the same limb, after 14 days we also record a delay of blood flow recovery. Similarly, in skin wounds we observe a delay of the healing in AdGRK5-NT-treated rats that associates with an impairment in the production of NFκB-dependent chemokine MCP-1. Our in vivo and in vitro data are therefore in agreement showing the regulatory effect of GRK5-NT on NFκB. It is interesting to note that upregulation of cellular GRK5 levels can be found in several conditions: Hypertension, for instance, presents increased GRK5 levels at the vasculature and impaired neoangiogenesis to chronic ischemia (45). Acute GRK5 upregulation to levels comparable with what we achieved in our setups and impairment of NFκB-dependent responses, such as cytokine production and chemotaxis, have been observed in neutrophils from patients with sepsis (46). Our data, therefore, open the field of investigation of GRK5's role in the regulation of NFκB-dependent phenotypes in vivo. Given the widespread distribution of GRK5 within the immunological system, it is possible to predict a role of GRK5 beyond angiogenesis and tissue repair. Indeed, in animals with targeted deletion of GRKs, the progression of various acute and chronic inflammatory disorders, including autoimmunity and allergy, is profoundly affected (47).

Materials and Methods

Cell Culture, GRK5 Gene Silencing, Apoptosis Analysis.

For cell culture, BAEC and HEK293 cells were used. The siRNA sequence against human GRK5 and the gene-silencing procedure have been described previously (48). Apoptosis was analyzed by Western blot with anti-cleaved caspase 3 antibody and by an Annexin-V-FLUOS staining kit (Roche).

Assays.

For overlay assay, GRK5, GRK2, MEK-I, and IκBα purified proteins were subjected to SDS/PAGE. The membrane was incubated with IκBα purified protein in binding buffer. For luciferase assay, cells were transfected with plasmid expression vectors coding for a κB-luciferase reporter and β-galactosidase and lysates analyzed by a luciferase assay system. For electrophoretic mobility shift assay, nuclear extracts from BAEC were subjected to electrophoresis in 8% nondenaturing polyacrilamide gels. Each gel was dried and subjected to autoradiography. For phosphorylation assay, purified GRK5 and GRK2 were incubated with Iκβα in binding buffer plus [γ-³²P]ATP; reactions were electrophoresed, and the gel was dried and subject to autoradiography. Migration and Matrigel assays were performed as previously described (49).

Northern Blot.

For Northern blot, total RNA from BAEC or from the hindlimbs of GRK5-NT and control rats was electrophoresed and transferred on nylon membrane. After hybridization, TNFα was evaluated by autoradiography.

In Vivo Study and Statistical Analysis.

Experiments were carried out in accordance with Federico II University guidelines on 12-week-old normotensive wistar Kyoto (WKY) male rats (n = 10), which had access to food and water ad libitum. The hindlimb ischemia, digital angiographies, and blood flow determination were performed as described previously (29). Skin wounds were made by excising the dorsal skin. Wound tissues were treated with pluronic gel containing AD-GRK5NT or AdEmpty, excised, and fixed for histology and immunohistochemistry. All values are presented as ±SEM. Two-way ANOVA was performed to compare the different parameters among the different groups. A significance level of P < 0.05 was assumed for all statistical evaluations. Statistics were computed with GraphPad Prism software. Extended details of experimental procedures are provided in SI Methods.