Caveolin-1 scaffolding domain residue phenylalanine 92 modulates Akt signaling

European Journal of Pharmacology 766 (2015) 46–55

Contents lists available at ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Caveolin-1 scaffolding domain residue phenylalanine 92 modulates Akt signaling Andy E. Trane a,b, Matti A. Hiob c, Tanya Uy a, Dmitri Pavlov a,b, Pascal Bernatchez a,b,n a

Centre for Heart Lung Innovation, St. Paul's Hospital, Canada Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Cananda c School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 June 2015 Received in revised form 18 September 2015 Accepted 21 September 2015 Available online 25 September 2015

Caveolin-1 (Cav-1), the homo-oligomeric coat protein of cholesterol-rich caveolae signalosomes, regulates signaling proteins including endothelial nitric oxide synthase (eNOS). The Cav-1 scaffolding domain (a.a. 82-101) inhibits activated eNOS from producing vascular protective nitric oxide (NO), an enzymatic process involving trafficking and phosphorylation. However, we demonstrated that Cav-1 proteins and peptides bearing F92A substitution (CAVF92A) could promote cardioprotective NO, most likely by preventing inhibition of eNOS by Cav-1. Herein, we showed that wild-type CAV sequence could, similar to CAVF92A, stimulate basal NO release, indicating a need to better characterize the importance of F92 in the regulation of eNOS by Cav-1/CAV. To reduce uptake sequence-associated effects, we conjugated a wild-type CAV derivative (CAVWT) or a F92A variant (CAVF92A) to antennapedia peptide (AP) or lipophilic myristic acid (Myr) and compared their effect on eNOS regulation in endothelial cells. We observed that both CAVWT and CAVF92A could increase basal NO release, although F92A substitution potentiates this response. We show that F92A substitution does not influence peptide uptake, endogenous Cav-1 oligomerization status and Cav-1 and eNOS distribution to cholesterol-enriched subcellular fractions. Instead, F92A substitution in CAVWT influences Akt activation and downstream eNOS phosphorylation status. Furthermore, we show that the cell permeabilization sequence could alter subcellular localization of endogenous proteins, an unexpected way to target different protein signaling cascades. Taken together, this suggests that we have identified the basis for two different pharmacophores to promote NO release; furthermore, there is a need to better characterize the effect of uptake sequences on the cellular trafficking of pharmacophores. & 2015 Elsevier B.V. All rights reserved.

Keywords: Endothelial nitric oxide synthase Caveolin-1 scaffolding domain Nitric oxide Antennapedia peptide Myristic acid

1. Introduction Caveolae are physical invaginations of the plasma membrane that spatially regulate signal transduction (Gratton et al., 2004; Parton and Pozo, 2013). The main coat protein of caveolae, caveolin-1 (Cav-1), is a 25 kDa protein with a tendency to form homo-oligomeric complexes exceeding 250 kDa (Fernandez et al., 2002; Sargiocomo et al., 1995); furthermore, it directly regulates a host of caveolae client proteins, such as G-protein coupled receptors, protein kinase A, adenylyl cyclase and endothelial nitric oxide (eNOS) (Babak et al., 1999; Garcia-Cardena et al., 1997; Gratton et al., 2004; Hagiwara et al., 2009), through a 20 amino acid sequence known as the Cav-1 scaffolding domain (a.a. 82101). Given the functional significance of Cav-1, many studies have n Correspondence to: Centre for Heart & Lung Health, St. Paul’s Hospital, James Hogg Research Centre, 1081 Burrard st, room 166, Vancouver, BC, Canada V6Z 1Y6. Fax: þ1 604 806 9274. E-mail address: [email protected] (P. Bernatchez).

http://dx.doi.org/10.1016/j.ejphar.2015.09.033 0014-2999/& 2015 Elsevier B.V. All rights reserved.

relied on cell permeable scaffolding domain-derived peptides to understand Cav-1-mediated regulatory events across a plethora of areas, from microvessel permeability to fibroblast regulation (Jasmin et al., 2006; Reese et al., 2013; Schmitz et al., 2011; Zhu et al., 2004); similarly, many have discussed the therapeutic implications for this class of peptides in a variety of disease settings such as pulmonary hypertension, hypertension, and inflammation (Bernatchez et al., 2011; Bucci et al., 2000; Jasmin et al., 2006). A major client protein of Cav-1 is eNOS, which produces nitric oxide (NO), a critical regulator of vascular homeostasis. NO is involved in atheroslerosis, hypertension, stroke, angiogenesis, and diabetes, amongst a host of other disease states (Cooke and Losordo, 2002; Huang et al., 1995; Napoli and Ignarro, 2001; Williams et al., 1996). While many factors can contribute to eNOS regulation, including cellular localization (Fulton et al., 2002) and nearby proteins (e.g. Akt, calmodulin and HSP 90 (Sessa, 2004)), eNOS inhibition is chiefly regulated by Cav-1 via a direct protein– protein interaction. Previously, we demonstrated that eNOS inhibition could be separated from binding, and that inhibition was

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

mediated by scaffolding domain residue F92 (Bernatchez et al., 2011), whereas binding was mediated by the binding site domain of the scaffolding domain (a.a. 90-99; CAV). Such separation of eNOS binding versus inhibition by Cav-1 allowed the use of alanine substituted F92A Cav-1 protein and peptides that bind eNOS without inhibiting its activity, which increase NO release due to attenuated endogenous Cav-1 inhibitory effect (Trane et al., 2014). Thus, we considered CAVF92A a potential pharmacophore for the development of peptide-protein inhibitors to induce NO release. However, given the promiscuous nature of the scaffolding domain, it is conceivable that other relevant signaling cascades may be disrupted by the F92A substitution. For example, an F92A mutation-containing peptide was previously shown to be better at inducing ERK phosphorylation in fibroblasts versus its wild type counterpart (Reese et al., 2013). Hence, we sought to investigate whether the mutation of F92 into alanine could unexpectedly alter eNOS activation indirectly, via changing the subcellular distribution of Cav-1 or eNOS, or directly, such as changing relevant signaling cascades. Herein, we report that CAV-derived peptides, with or without an intact F92 residue, were able to promote basal NO release, indicating that F92 may have functions outside of direct eNOS inhibition. To confirm this, we fused CAVWT and CAVF92A to either antennapedia (AP) or myristic acid (Myr) uptake sequences, allowing us to mitigate carrier-dependent subcellular trafficking differences. We found that both Myr- and AP-conjugated peptides have different effects on Cav-1/eNOS co-localization, indicating that there are some carrier-associated differences present. However, the unique ability of CAVWT, and the inability of CAVF92A, to activate Akt appears to be regulated by the F92 residue. Hence, this shows that F92 is critical to the regulation of Akt-dependent signaling, and that various cell uptake sequences have unique cell distribution properties.

47

CAVWT or CAVF92A peptides conjugated to either AP or Myr (10 mM). Cells were imaged on a Leica inverted microscope with a confocal scanner and peptide uptake was analyzed using Volocity software (PerkinElmer, Massachusetts). 2.4. Immunoblotting BAECs were treated with peptides (10 mM) for the specified duration. Cells were lysed in a buffer containing (50 mM Tris–HCl, 1% NP-40, 0.1% SDS, 0.1% deoxycholic acid, 0.1 mM EDTA, 0.1 mM EGTA, 5 mM NaF, 2 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF and Sigma protease inhibitor cocktail). Protein concentration was measured using a Bio-Rad DC assay, total proteins were separated on a 7.5%/12% split gradient SDSPAGE gel and transferred on to a nitrocellulose membrane as previously described. Membranes were probed with rabbit antiphospho-Akt-473 (Cell Signaling, Massachusetts), rabbit anti-total Akt (Cell Signaling), mouse total eNOS (BD transduction) and rabbit anti-phospho-eNOS-1179 (Cell Signaling). HSP-90 (BD Transduction) was used as a loading control. Blots were scanned using a LI-COR infrared imaging system to allow linear quantification of fluorescent signal. 2.5. Co-localization imaging

2. Materials and methods

BAECs were treated with peptides for 4 h before being fixed in 10% neutral buffered formalin. Cells were permabilized with 0.1% Triton X-100 and blocked with PBS containing 2.5% normal goat serum and 0.1% low IgG bovine serum albumin (Millapore). To identify proteins of interest, BAECs were incubated with rabbit anti-Caveolin-1 (Santa Cruz Biotech, California) and mouse antieNOS (BD Transduction), followed by goat anti-rabbit Alexa 594 and goat anti-mouse Alexa 488 (Molecular Probes, Invitrogen). Specificity was confirmed using a mouse and rabbit normal IgG (Santa Cruz Biotech) control. Slides were imaged on a Leica inverted microscope as described above.

2.1. Cell culture

2.6. Sucrose fractionation

Bovine aortic endothelial cells (BAECs) were grown using Dulbecco's Modified Eagle Medium (Invitrogen) containing 5% fetal bovine serum, 100 units of penicillin and 0.1 mg streptomycin in a 37 °C incubator with 7% CO2. Cells were used between passages 3 and 10.

Cholesterol-enriched membrane fractions were isolated as previously described (Bernatchez et al., 2007). Confluent cells were serum starved for 24 h, followed by treatment with the indicated peptides for 6 h. Cells were then lysed in a NaCO3-based (500 mM) buffer containing a protease inhibitor cocktail (Sigma Aldrich) and sonicated (Bernatchez et al., 2007). The lysate was combined with a 90% sucrose solution and overlayed with 30% and 5% sucrose layers successively. All sucrose solutions were prepared in MBS (25 mM MES, 150 mM NaCl, pH 6.5). Samples were centrifuged for 18 h at 140,000g using a SW 41 Ti Rotor and fractions were collected top down.

2.2. Nitric oxide analysis Peptides were sourced from Elim Biopharm (California) while myristic acid was purchased from Sigma Aldrich (Missouri). BAECs were incubated with the stated compounds (10 mM) for 6 h in serum free DMEM, following which the media was collected. For Akt studies, cells were pre-incubated with Wortmannin (1 mM) for 1 h before peptide treatment. Analysis of NO levels was performed as previously described using our Sievers NO analyzer (Trane et al., 2014). The area under the curve for the media responses were compared against a standard curve created using known concentrations of sodium nitrite. 2.3. Peptide uptake BAECs were transiently transfected in 35 mm glass-bottom plates (MatTek, Massachusetts) with YFP-tagged Cav-1, a kind gift from Dr. Richard Minshall, using Lipofectamine 2000 (Invitrogen, Massachusetts) followed by 2 days of incubation for plasmid expression. Cells were then blocked with unconjugated peptide for 1 h before being incubated with Cy-5 fluorophore conjugated

2.7. Velocity gradient fractionation Confluent cells were serum starved for 24 h, followed by treatment with the indicated peptides for 6 h. Subsequently, cells were lysed in MBS containing 60 mM octyl-β-glycopyranoside (Sigma Aldrich), sonicated and loaded on top of a 5–45% linear sucrose gradient. Samples were centrifuged for 18 h at 140,000g using a SW 41 Ti Rotor and fractions were collected top down. 2.8. Data and statistical analysis NO release and peptide uptake results were analyzed using two-way ANOVA with a Dunnett's post-hoc analysis to identify between-group differences. Cav-1 and eNOS localization results were analyzed with one-way ANOVA.

48

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

3. Results 3.1. Both CAVWT and CAVF92A can increase NO release independent of uptake sequence Having identified that Cav-1 scaffolding domain residues 90-99 were the main contributors to eNOS binding (Trane et al., 2014) and that binding was not dependent on F92 (which is considered the main inhibitory residue (Bernatchez et al., 2005)), we sought to better characterize the importance of F92 in the context of the binding sequence (90-99), rather than the entire Cav-1 scaffolding domain (82-101) or full-length Cav-1 protein (Bernatchez et al., 2011; Trane et al., 2014). Therefore cell-permeable CAV 90-99 peptides were synthesized and tested for NO release. In the absence of a cargo sequence, the cell permeable vehicles AP and Myr could not solicit an NO response from cultured BAEC that endogenously express eNOS and Cav-1 (Fig. 1A). However, AP- and Myr-CAVF92A were able to promote basal NO release, although Myr-CAVF92A induced significantly higher levels of NO production (2145 pmol NO/mL) in comparison to the AP variant (367 pmol). Unexpectedly, both AP- and Myr-CAVWT peptides were also able to increase basal, unstimulated NO release, although the level of NO was not significantly different between the two peptides (1193 vs 788 pmol NO/mL respectively; Fig. 1A). This suggests that the previously identified eNOS binding site sequence of Cav-1 with the F92A substitution, CAVF92A, can trigger the release of far greater amounts of NO that CAVWT, a sequence generally associated with eNOS inhibition, although CAVF92A requires Myr-based cellular

uptake for high levels of efficacy. We then performed a peptide uptake study to ensure that the difference in NO release between the CAVWT and CAVF92A peptides used above were not attributable to varying rates of uptake. We found that the single point substitution itself (CAVWT vs CAVF92A) did not have an effect on uptake as determined by total fluorescence intensity in live BAEC, regardless of which uptake sequence the cargo was conjugated to (Fig. 1B); however, AP-conjugated peptides promoted greater levels of uptake compared to Myrconjugated peptides. Taken together with the dogma that the fulllength Cav-1 scaffolding sequence is well known to have an inhibitory effect on activated eNOS, this suggests that not only do sub-segments of the full-length Cav-1 scaffolding domain (82-101) have different regulatory effects on eNOS, but also that these effects could be modulated by other differences in uptake sequence characteristics. 3.2. Increases in NO release are not attributable to changes in Cav-1 oligomerization Being that the sequence was derived from the Cav-1 scaffolding domain, known to be part of the oligomerization domain (Sargiacomo et al., 1995), we wanted to confirm that the increase in NO release observed in Fig. 1A was not attributable to Cav-1 oligomers destabilization, which could have profound implications for caveolae function, general cell signaling and eNOS activation. To confirm this, we performed velocity gradient fractionation to assess for molecular weight changes. We previously calibrated the separation by molecular weight for a range of MW markers including carbonate anhydrase, BSA, catalase, and thyroglobulin (Bernatchez et al., 2011); this was further confirmed by probing for both Akt (60 kDa) and eNOS (140 kDa), in addition to Cav-1 (25 kDa; Fig. 2A). Akt appeared in fractions 2–4, whereas eNOS starts appearing in fraction 4, as would be expected due to their size difference. In contrast, Cav-1 begins appearing in fraction 4 and continues up to fraction 12, indicating the existence of high molecular weight oligomers. Following treatment with either AP or Myr, alone or fused to a cargo sequence (CAVWT or CAVF92A), we did not observe any qualitative changes in Cav-1 oligomer distribution (Fig. 2B and C). We subsequently performed densitometric quantification of Cav-1 oligomer distribution (Fig. 2D and E), which clearly demonstrated that neither uptake sequence, nor F92A substitution, affected the oligomerization status of Cav-1, with the bulk of the Cav-1 protein appearing in fractions 4 and 5. 3.3. Subcellular targeting of Cav-1 and eNOS to cholesterol-rich membrane microdomains is not affected by F92A substitution

Fig. 1. CAVWT and CAVF92A peptides can induce NO release. (A) Level of nitrite (pmol/mL), expressed as mean7 S.E.M. (n ¼6–7), induced by AP- (white bars) or Myr- (black bars) conjugated peptides. No significant difference was observed between the CAVWT peptides; in contrast, Myr-CAVF92A promoted significantly greater levels of nitrite compared to AP-CAVF92A (*Po 0.05). (B) Relative levels of AP- and Myr-conjugated CAVWT and CAVF92A taken up by BAECs (mean7 S.E.M.; n¼ 5).

As no changes were observed in Cav-1 oligomerization, we proceeded to investigate whether uptake sequence or F92 substitution caused changes in the cellular localization of target proteins, Cav-1 and eNOS, to cholesterol rich membranes. This stems from the observation that proteins such as eNOS are known to behave differently following targeting to Golgi or cholesterol-rich domains (Fulton et al., 2002). To confirm targeting to cholesterol rich regions, we used the sucrose fractionation assay as previously published (Bernatchez et al., 2011). As demonstrated in Fig. 3A, HSP 90, a well-known cytosolic marker, was found in fractions 8– 12; in contrast, Cav-1, which has been used extensively as a marker for cholesterol rich membrane, was found predominantly in fractions 2 and 3. Lastly, eNOS, which is known to distribute between both cholesterol- and non-cholesterol rich regions, was indeed found to be associated with both areas. Treatment of cells with CAVWT and CAVF92A peptides, regardless of conjugation to AP (Fig. 3B) or Myr (Fig. 3C), did not change Cav-1 targeting to cholesterol-rich fractions, with the bulk of the oligomers showing up

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

49

Oligomers - Increasing MW Fraction:

1

12

eNOS Akt Cav-1

Fraction:

1

12

1

12

AP AP-CAVWT AP-CAVF92A

Fraction: Myr Myr-CAVWT Myr-CAVF92A

Fig. 2. CAVWT and CAVF92A peptides did not affect Cav-1 oligomerization. (A) Representative immunoblot indicating the distribution of eNOS, Akt and Cav-1 following velocity gradient centrifugation. Molecular weight increases from left to right (indicated by gradient bar above figure). (B and C) Representative immunoblots demonstrating the distribution of Cav-1 oligomers following AP- or Myr-conjugated peptide treatment, respectively. (D and E) Graphical representation (mean 7 S.E.M.; n¼ 3) of the percent of total Cav-1 in each fraction following treatment by peptides conjugated to either AP or Myr respectively.

in fractions 2–6. This was verified by densitometric quantification of Cav-1 in each fraction following peptide treatment (Fig. 3D and E). Similarly, we sought to confirm whether eNOS distribution was altered by F92A substitution following peptide treatment. Compared to wild type CAV, the F92A substitution had little bearing on the cholesterol-rich microdomains targeting of eNOS protein following peptide treatment (Fig. 4 A–D), regardless of whether it was conjugated to AP or Myr. On the other hand, when compared to control AP or Myr stimulation, the CAV peptides triggered a slight shift of eNOS from the buoyant fractions to the non-lipid fractions, which is a known outcome of eNOS activation, regardless of F92 inactivation, (Sanchez et al., 2006). This suggests that, indeed, the CAVWT, sequence can change underlying mechanisms of eNOS regulation to promote NO release and that this triggers a shift of eNOS outside of caveolae/lipid rafts. 3.4. Co-localization of Cav-1 and eNOS is influenced by cell uptake sequence but not F92A substitution Since we observed a trend towards eNOS redistribution in

response to CAVWT/CAVF92A stimulation, this indicated that eNOS and Cav-1 co-localization may be altered. As such, we sought to verify this by investigating Cav-1 and eNOS distribution within the cell through fixed-cell immunofluorescence following peptide treatment. We found no qualitative differences in eNOS and Cav-1 staining between AP, AP-CAVwt and AP-CAVF92A-treated cells, and no change in eNOS/Cav-1 co-localization as analyzed using a Pearson’s correlation analysis (Fig. 5A). Similarly Myr, Myr-CAVWT and Myr-CAVF92A treatments did not cause changes in eNOS and Cav-1 staining patterns when compared to each other (Fig. 5B). In contrast, within the confocal images, we do observe a qualitative difference in Cav-1 distribution depending on whether the cells were treated with AP- or Myr-conjugated sequences. More specifically, AP-based peptides tended to have a more membrane centric pattern, while Myr-based sequences induced cyotoplasmic Cav-1 staining. This difference in co-localization pattern may help to explain the difference between AP- and Myr-CAVF92A induced NO release levels (Fig. 1). For example Cav-1 trafficking may contribute to the maintenance of active signaling complexes different from that associated with plasma membrane. However, it still does

50

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

Cholesterol-rich

Fraction:

1

bulk proteins

12

eNOS HSP 90 Cav-1

Cholesterol-rich

Fraction:

bulk proteins

1

12

AP

WB: Cav-1

AP-CAVWT AP-CAVF92A

Cholesterol-rich

Fraction:

WB: Cav-1

bulk proteins

1

12

Myr Myr-CAVWT Myr-CAVF92A

Fig. 3. CAVWT and CAVF92A peptides did not affect Cav-1 distribution to lipid rich areas in endothelial cells. (A) Representative immunoblot indicating the distribution of eNOS, HSP-90 and Cav-1 to the buoyant lipid raft (1-7) and cytosolic fractions (8-12) following sucrose fractionation. (B and C) Representative immunoblots demonstrating the distribution of Cav-1 following AP- or Myr-conjugated peptide treatment, respectively. (D and E) Graphical representation (mean 7 S.E.M.; n ¼3) of the percent of total Cav-1 associated with each individual fraction following treatment by peptides conjugated to either AP (D) or Myr (E) respectively.

not address the mechanistic difference in NO release between CAVWT and CAVF92A. 3.5. F92A substitution reduces CAVWT-mediated Akt activation but not NO release Thus far, our results indicated that F92A substitution does not alter any of the basic cellular biochemical properties of eNOS and Cav-1; therefore, we sought to assess for changes with respect to intracellular signaling instead. Interestingly, we demonstrated that there was an increase in phosphorylation of Akt, one of the most prominent activators of eNOS signaling, following treatment with either AP- or Myr-CAVWT peptides, which was mimicked to a lesser extent by their CAVF92A counterparts (Fig. 6A and B). As would be expected, we also simultaneously observed an increase in the phosphorylation state of eNOS at 1179D, which is important for the full activation of eNOS activity (Fulton et al., 1999). To confirm that Akt signaling does indeed drive NO production by CAVWT, we treated endothelial cells with wortmannin (1 mM), an

irreversible inhibitor of the phosphoinositide 3-kinase (PI3k)/Akt signaling pathway, and found that AP-and Myr-CAVWT-induced NO release was significantly reduced by 46% and 53% respectively (Fig. 6C and D). In contrast, minimal effects were observed on APand Myr-CAVF92A-induced NO release, indicating that F92 plays a critical role in the regulation of Akt signaling.

4. Discussion Cav-1, via its scaffolding domain, is a critical regulator of a plethora of proteins including eNOS, which is essential for cardiovascular homeostasis. Due to its significant vascular role in health and disease, there is value in exploring novel approaches to promoting eNOS-mediated NO release. We propose that peptides derived from Cav-1 could be used to this end. Interestingly, we found that, in the context of unstimulated settings, both CAVWT and CAVF92A could promote NO release, with initial evidence pointing at differing mechanisms. In order to mitigate uptake

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

Cholesterol-rich

WB: eNOS

Fraction:

51

Bulk proteins

1

12

AP AP-CAVWT AP-CAVF92A

Cholesterol-rich

WB: eNOS

Fraction:

Bulk proteins

1

12

Myr Myr-CAVWT Myr-CAVF92A

Fig. 4. CAVWT and CAVF92A peptides did not affect Cav-1 eNOS distribution to lipid rich areas in endothelial cells. (A and B) Representative immunoblots demonstrating the distribution of eNOS following AP- or Myr-conjugated peptide treatment, respectively. (C and D) Graphical representation (mean 7 S.E.M.; n¼ 3) of the percent of total eNOS associated with each individual fraction following treatment by peptides conjugated to either AP (C) or Myr (D) respectively.

sequence-driven biases, we choose to perform the study using two distinctly different uptake methods, which allowed us to better assay for the role of F92 in eNOS regulation. Since the peptide sequences used originated from the oligomerization domain, defined as residues 61-101 of the Cav-1 (Sargiacomo et al., 1995), there were initial concerns about disrupting Cav-1 oligomers. However, as Cav-1 complex sizes were unchanged, it suggests that within the time frame assessed changes in oligomerization status was not a major contributor to NO production, as would be expected, since Cav-1 oligomeric complexes are known to be stable (Tagawa et al., 2005). Furthermore, sub-cellular targeting of protein did not seem to be affected by F92A substitution. In contrast, F92A substitution played a unique role in uncoupling NO release from Akt activation. Taken together, our results indicate that that the F92 residue has a significant impact on basal NO regulation via Akt signaling, independent of the uptake sequence utilized. 4.1. Role of the permeabilization sequence Since Cav-1-derived peptides have been typically conjugated to cell permeable antennapedia peptide (AP), a cellular internalization sequence derived from the third helix of the DNA binding domain of antennapedia (Derossi et al., 1996), we addressed potential uptake and sub-cellular trafficking-related issues by comparing AP-conjugated peptides against an additional carrier. Of interest is myristic acid (Myr), a 14 carbon lipid chain, which has been used to deliver agents such as PKC-α pseudosubstrate peptides (Bergman et al., 1997) and gadolinium-DOTA, a MRI contrast agent (Sturzu et al., 2009) in to cells. Myristoylation is a naturally occurring form of post-translational modification for proteins such as eNOS, which helps target it to the plasma membrane (Shaul et al., 1996). Furthermore, myristoylation of peptides is thought to

promote its association with the plasma membrane (Pham et al., 2004), making it an appealing comparator to AP, given the site of eNOS/Cav-1 interaction. Different cell lines have been known to exhibit preference for specific uptake carriers, as shown in a study comparing Myr to TAT (a cell-penetrating peptide sequence derived from HIV-1 (Vivès et al., 1997)) which found that lymphocytes more readily took up the Myr-conjugated peptide sequence (Nelson et al., 2007). In contrast, our study demonstrated that uptake of AP-based peptides was greater than Myr-based peptides. This may be the result of the uptake mechanisms, as protein based uptake sequences, such as AP and TAT, have been conjectured to be uptaken by lipid raft dependent, clathrin-independent endocytosis (Jones et al., 2005) and macropinocytosis (Wadia et al., 2004) respectively, whilst, lipidated peptides are thought to associate with the membrane and enter the cell via flip-flop exchange (Eisele et al., 2001; Nelson et al., 2007). The membrane targeted entry method of lapidated peptides may allow it to better target membrane specific processes (i.e. Cav-1/eNOS interactions), which may explain why lower levels of Myr-based peptide uptake could generate such robust responses and contribute to some of the carrier dependent effects observed, which could lead to substantial differences in therapeutic outcomes. This suggests that Myr-conjugation may be a better alternative than the traditionally utilized AP-conjugation for investigating Cav-1 based regulation of cellular mechanisms and therapies. 4.2. Characterization of critical residue F92 There have been many studies investigating the scaffolding domain of Cav-1 to date; however, such studies tend to study the impact of several residues simultaneously, as part of what is known as the Cav-1 binding motif (Couet et al., 1997). Another

52

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

eNOS

Cav-1

Merge

eNOS

Cav-1

Merge

AP

APCAVWT

APCAVF92A

Myr

MyrCAVWT

MyrCAVF92A

Fig. 5. Cell permeabilization sequence alters Cav-1 distribution and Cav-1/eNOS co-localization (A) Representative confocal images of formaldehyde fixed endothelial cells demonstrating eNOS (green) and Cav-1 (red) co-localization following treatment with AP and AP-conjugated peptides. Higher levels of plasma membrane association was observed (white arrow); however, all treatment conditions (AP, AP-CAVWT and AP-CAVF92A) had no difference in the level of eNOS/Cav-1 co-localization. (B) Equivalent images for endothelial cells treated with Myr-conjugated peptides, showing a more cytoplasmic staining pattern for Cav-1. Similarly, Pearson's correlation indicated that the degree of co-localization between Cav-1 and eNOS following treatment Myr-conjugated peptide treatment did not produce a change in the leve of co-localization. N ¼ 6 and mean 7S.E.M for both experiments.

common approach is to perform a F92A/V94A substitution, which has been used for a range of studies including insulin-mediated Cav-1 phosphorylation (Kimura et al., 2002), negative regulation of raft-dependent endocytosis (Lajoie et al., 2009), Elk-1 signaling in adipose cells (Nystrom et al., 1999) and epidermal growth factorinduced p-ERK signaling (Lajoie et al., 2007). However, such studies would not be able to attribute the observed effects to specific residues. Furthermore, as shown by our previous investigations

(Bernatchez et al., 2011; Bernatchez et al., 2005; Trane et al., 2014), and also in this study, a one point mutation could have large implications for eNOS regulation. Similarly, another group studying monocytes and fibroblasts observed that a single point mutation, F92A, could have a profound effect on cellular events. This suggests that there is a need to focus on specific residues to develop a true understanding of how the Cav-1 scaffolding domain is regulating cellular biology and its potential therapeutic implications,

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

p-eNOS

p-eNOS

eNOS

eNOS

HSP 90

HSP 90

p-AKT

p-AKT

AKT

AKT

53

Fig. 6. CAVWT-induced NO release is Akt dependent. (A and B) Representative blot comparing changes in Akt and eNOS phosphorylation states in AP- (AP, AP-CAVWT or APCAVF92A) or Myr-peptide (Myr, Myr-CAVWT or Myr-CAVF92A) treated endothelial cells. HSP 90 was utilized as a loading control. Densitometry data shown below blots are for a single observation; data expressed as a percentage of the highest ratio for either phosphorylated/total eNOS or Akt. The peptides were able to promote phosphorylation of Akt (Obervations repeated three times). (C) AP-, AP-CAVWT- and AP CAVF92A-induced nitrite (pmol/mL) in endothelial cells in the absence (white) or presence (black) of wortmannin (1 μM), an inhibitor of Akt activation (mean 7 S.E.M.; n¼ 4; *Po 0.05). (D) Equivalent study using Myr-conjugated peptides (mean 7S.E.M.; n ¼4; *Po 0.05) in the absence (white) or presence (black) of wortmannin.

so as to minimize or alter the effect profile. 4.3. Peptide-induced redistribution of caveolin-1 Protein distribution is well known to alter function. As such, we hypothesized that there could be a change in localization of either eNOS or Cav-1 induced by the F92A substitution. We first investigated eNOS, which is differentially regulated based on its localization in the golgi or plasma membrane (Fulton et al., 2004);

however, our sucrose fractionation results suggested that this was not a major contributor, which was qualitatively supported by our immunofluorescence study. In contrast, a noticeable shift was observed in the Cav-1 distribution. Interestingly, AP-peptide treatment induced the re-distribution of Cav-1 to the plasma membrane; in comparison, Myr-peptides produced a diffused staining pattern. However, the sucrose fractionation profiles looked similar between the AP- and Myr-treated cells, suggesting that the cytosolic Cav-1 fractionated to cholesterol rich fractions.

54

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

Indeed, others have shown that cytosolic Cav-1 can fractionate to cholesterol-rich fractions (Ito et al., 2002). In addition, as Cav-1 at the plasma membrane is known to drive caveolae formation, redistribution of Cav-1 could have profound consequences on downstream events. Even if caveolae formation was not affected, Cav-1 is a regulator of a plethora of proteins; hence, it is likely that other aspects of cell signaling could be perturbed. This suggests that Myr- and AP-based peptides may be utilized to study the functional significance of specific mutations on cargo sequences in different regions of the cell, which could help with the development of targeted therapies. However, in the context of our study, by combining observations from both carriers, we can mitigate some of this carrier driven response while trying to isolate the effect of F92 on cellular processes. 4.4. Akt regulation by CAV F92 As basic biochemical properties of both eNOS and Cav-1 were not affected by F92A substitution, we decided to investigate signaling processes. Cav-1 has been shown to promote PI3k/Akt activity, known to drive eNOS activity by promoting phosphorylation of S1179 (Dimmeler et al., 1999; Fulton et al., 1999), in events such as type I procollagen expression (Kim et al., 2008), cancer cell migration (Chanvorachote et al., 2014), cell survival and vascular mechanotransduction (Sedding et al., 2005) through its scaffolding domain (Li et al., 2003). Similarly, in our study, we observed that the wild type CAVWT sequence was able to increase pAkt and drive NO production. This was further confirmed by the significant decrease in NO release following treatment with wortmannin, a well-known irreversible inhibitor of PI3k. In contrast, the substitution of F92 in CAVWT removed the sensitivity to wortmannin, suggesting that this residue plays an important role in PI3k/Akt signaling regulation. This is supported by findings from another group that suggested F92A substitution in cavtratin, the full length scaffolding domain peptide, led to increased pERK and pMEK, indicating that a single residue could have profound implications on scaffolding domain peptide-mediated signaling. Lastly, a small, but insignificant, decrease in NO production was observed in MyrCAVF92A treated cells following wortmannin incubation, which may be due to the nature of Myr having the potential to promote Akt phosphorylation (Krotova et al., 2006), or residual regulatory effects of the cargo sequence.

5. Conclusion Cav-1 is not only important for its role in generating caveolae, but also for its ability to interact with and regulate caveolae-associated proteins. As such, many researchers have utilized Cav-1 derived peptides for a plethora of mechanistic studies. This study highlights two points of interest. Firstly, F92 is involved not only in eNOS regulation, but potentially Akt signaling as well. Secondly, the cell permeabilization sequence has to be chosen with care, as it could have a significant impact on Cav-1 distribution and potentially caveolae-mediated signaling and serve as a confounding variable in mechanistic and therapeutics studies, as it could significantly potentiate or reduce the response, as well as alter intracellular interactions.

Acknowledgments We would like to acknowledge CIHR, Heart and Stroke Foundation of Canada, British Columbia & Yukon (AT, PB) Canada Foundation for Innovation, British Columbia Knowledge

Development Fund and Michael Smith Foundation for Health Research for salary support.

References Babak, R., Rubin, C.S., Lisanti, M.P., 1999. Regulation of cAMP-mediated signal transduction via interaction of caveolins with the catalytic subunit of protein kinase A. J. Biol. Chem. 274, 26353–26360. Bergman, P., Gravitt, K., Ward, N., Beltran, P., Gupta, K., O’Brian, C., 1997. Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-alpha pseudosubstrate peptides through a P-glycoprotein-independent mechanism. Invest. New Drugs 15, 311–318. Bernatchez, P., Sharma, A., Bauer, P.M., Marin, E., Sessa, W.C., 2011. A Noninhibitory mutant of the Caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice. J. Clin. Invest. 121, 3747–3755. Bernatchez, P.N., Acevedo, L., Fernandez-Hernando, C., Murata, T., Chalouni, C., Kim, J., Erdjument-Bromage, H., Shah, V., Gratton, J.-P., McNally, E.M., Tempst, P., Sessa, W.C., 2007. Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function. J. Biol. Chem. 282, 30745–30753. Bernatchez, P.N., Bauer, P.M., Yu, J., Prendergast, J.S., He, P., Sessa, W.C., 2005. Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell permeable peptides. Proc. Natl. Acad. Sci. USA 102, 761–766. Bucci, M., Gratton, J.-P., Rudic, R.D., Acevedo, L., Roviezzo, F., Cirino, G., Sessa, W.C., 2000. In vivo delivery of the Caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation. Nat. Med. 6, 1362–1367. Chanvorachote, P., Chunhacha, P., Pongrakhananon, V., 2014. Caveolin-1 Induces lamellipodia formation Via an Akt-dependent pathway. Cell Int. 14, 52. Cooke, J.P., Losordo, D.W., 2002. Nitric oxide and angiogenesis. Circulation 105, 2133–2135. Couet, J., Li, S., Okamoto, T., Ikezu, T., Lisanti, M.P., 1997. Identification of peptide and protein ligands for the caveolin-scaffolding domain. J. Biol. Chem. 272, 6525–6533. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., Prochiantz, A., 1996. Cell internalization of the third helix of the antennapedia hoeodomain is receptor-independent. J. Biol. Chem. 271, 18188–18193. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., Zeiher, A.M., 1999. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605. Eisele, F., Kuhlmann, J., Waldmann, H., 2001. Synthesis and membrane-binding properties of a characteristic lipopeptide from the membrane-anchoring domain of influenza virus A hemagglutinin. Angew. Chem. Int. Ed. 40, 369–373. Fernandez, I., Ying, Y., Albanesi, J., Anderson, R.G.W., 2002. Mechanism of caveolin filment assembly. Proc. Natl. Acad. Sci. USA 99, 11193–11198. Fulton, D., Babbit, R., Zoellner, S., Fontana, J., Acevedo, L., McCabe, T.J., Iwakari, Y., Sessa, W.C., 2004. Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release. J. Biol. Chem. 279, 30349–30357. Fulton, D., Fontana, J., Sowa, G., Gratton, J.-P., Lin, M., Li, K.-X., Michell, B., Kemp, B.E., Rodman, D., Sessa, W.C., 2002. Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in golgi and plasma defines the existence of two pools of active enzyme. J. Biol. Chem. 277, 4277–4284. Fulton, D., Gratton, J.-P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F., Papapetropoulos, A., Sessa, W.C., 1999. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597–601. Garcia-Cardena, G., Martasek, P., Masters, B.S.S., Skidd, P.M., Couet, J., Li, S., Lisanti, M.P., Sessa, W.C., 1997. Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. J. Biol. Chem. 272, 25437–25440. Gratton, J.-P., Bernatchez, P., Sessa, W.C., 2004. Caveolae and caveolins in the cardiovascular system. Circ. Res. 94, 1408–1417. Hagiwara, M., Shirai, Y., Nomura, R., Sasaki, M., Kobayashi, K.-I., Tadokoro, T., Yamamoto, Y., 2009. Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction. Biochem. Biophys. Res. Commun. 378, 73–78. Huang, P.L., Huang, Z., Mashimo, H., Bloch, K.D., Moskowitz, M.A., Bevan, J.A., Fishman, M.C., 1995. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377, 239–242. Ito, J.-i, Nagayasu, Y., Kato, K., Sato, R., Yokoyama, S., 2002. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocyte. J. Biol. Chem. 277, 7929–7935. Jasmin, J.-F., Mercier, I., Jocelyn, D., Tanowitz, H.B., Lisanti, M.P., 2006. Short-term administration of a cell-permeable caveolin-1 peptide prevents the development of monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy. Circulation 114, 912–920. Jones, S.W., Christison, R., Bundell, K., Voyce, C.J., Brockbank, S.M.V., Newham, P., Lindsay, M.A., 2005. Characterisation of cell-penetrating peptide-mediated peptide delivery. Br. J. Pharmacol. 145, 1093–1102. Kim, S., Lee, Y., Seo, J.E., Cho, K.H., Chung, J.H., 2008. Caveolin-1 increases basal and TGF-β1-induced exprssion of type I procollagen through PI-3 kinase/Akt/mTOR pathway in human dermal fibroblasts. Cell Signal 20, 1313–1319. Kimura, A., Mora, S., Shigematsu, S., Pessin, J.E., Saltiel, A.R., 2002. The insulin receptor catalyzes the tyrosine phosphorylation of caveolin-1. J. Biol. Chem. 277, 30153–30158. Krotova, K., Hu, H., Xia, S.-L., Belayev, L., Patel, J.M., Block, E.R., Zharikov, S., 2006.

A.E. Trane et al. / European Journal of Pharmacology 766 (2015) 46–55

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation. Br. J. Pharmacol. 148, 732–740. Lajoie, P., Kojic, L.D., Nim, S., Li, L., Dennis, J.,W., Nabi, I.R., 2009. Caveolin-1 regulation of dynamin-dependent, raft-mediated endocytosis of cholera toxin-B sub-unit occurs independently of caveolae. J. Cell Mol. Med. 9B, 3218–3225. Lajoie, P., Patridge, E.A., Guay, G., Goetz, J.G., Pawling, J., Lagana, A., Joshi, B., Dennis, J.W., Nabi, I.R., 2007. Plasma membrane domain organization regulates EGFR signaling in tumor cells. J. Cell Biol. 179, 341–356. Li, L., Ren, C.H., Tahir, S.A., Ren, C., Thompson, T.C., 2003. Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. Mol. Cell Biol. 23, 9389–9404. Napoli, C., Ignarro, L.J., 2001. Nitric Oxide and Atherosclerosis. Nitric Oxide 5, 88–97. Nelson, A.R., Borland, L., Allbritton, N.L., Sims, C.E., 2007. Myristoyl-based transport of peptides into living cells. Biochemistry 46, 14771–14781. Nystrom, F.H., Chen, H., Cong, L.-N., Li, Y., Quon, M.J., 1999. Caveolin-1 interacts with the insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells. Mol. Endocrinol. 13, 2013–2024. Parton, R.G., Pozo, M.A., 2013. Caveolae as plasma membrane sensors, protectors and organizers. Nat. Rev. 14, 98–112. Pham, W., Kircher, M.F., Weissleder, R., Tung, C.-H., 2004. Enhancing membrane permeability by fatty acylation of oligoarginine peptides. ChemBioChem 5, 1148–1151. Reese, C., Dyer, S., Perry, B., Bonner, M., Oates, J., Hofbauer, A., Sessa, W.C., Bernatchez, P., Visconti, R.P., Zhang, J., Hatfield, C.M., Silver, R.M., Hoffman, S., Tourkina, E., 2013. Differential regulation of cell functions by CSD peptide subdomains. Respir. Res. 14, 1–18. Sanchez, F.A., Savalia, N.B., Duran, R.G., Lal, B.K., Boric, M.P., Duran, W.N., 2006. Functional significance of differential eNOS translocation. Am. J. Physiol. Heart Circ. Physiol. 291, H1058–H1064. Sargiacomo, M., Scherer, P.E., Tang, Z., Kubler, E., Song, K.S., Sanders, M.C., Lisanti, M. P., 1995. Oligomeric structure of caveolin: implications for caveolae membrane oligomerization. Proc. Natl. Acad. Sci. USA 92, 9407–9411. Sargiocomo, M., Scherer, P.E., Tang, Z., Kubler, E., Song, K.S., Sanders, M.C., Lisanti, M.P., 1995. Oligomeric struture of caveolin: implications for caveolae

55

membrane organization. Proc. Natl. Acad. Sci. USA 92, 9407–9411. Schmitz, M., Zerr, I., Althaus, H.H., 2011. Effect of cavtratin, a caveolin-1 scaffolding domain peptide on oligodendroglial signaling cascades. Cell Mol. Neurobiol. 31, 991–997. Sedding, D.G., Hermsen, J., Seay, U., Eickelberg, O., Kummer, W., Schewencke, C., Strasser, R.H., Tillmanns, H., Braun-Dullaeus, R.C., 2005. Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo. Circ. Res. 96, 635–642. Sessa, W.C., 2004. eNOS at a glance. J. Cell Sci. 117, 2427–2429. Shaul, P.W., Smart, E.J., Robinson, L.J., German, Z., Yuhanna, I.S., Ying, Y., Anderson, R.G.W., Michel, T., 1996. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J. Biol. Chem. 271, 6518–6522. Sturzu, A., Klose, U., Echner, H., Beck, A., Gharabaghi, A., Kalbacher, H., Heckl, S., 2009. Cellular uptake of cationic gadolinium-DOTA peptide conjugates with and without N-terminal myristoylation. Amino Acids 37, 249–255. Tagawa, A., Mezzacasa, A., Hayer, A., Longatti, A., Pelkmans, L., Helenius, A., 2005. Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered vesicular transporters. J. Cell Biol. 170, 769–779. Trane, A.E., Pavlov, D., Sharma, A., Saqib, U., Lau, K., Petegem, Fv, Minshall, R.D., Roman, L.J., Bernatchez, P., 2014. Deciphering the binding of caveolin-1 to client protein endothelial nitric oxide synthase (eNOS): scaffolding sub-domain identification, interaction modeling, and biological significance. J. Biol. Chem. 289, 13273–13283. Vivès, E., Brodin, P., Lebleu, B., 1997. A truncated HIV-1 tat protein basic domain rapidly translocated through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010–16017. Wadia, J.S., Stan, R.V., Dowdy, S.F., 2004. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315. Williams, S.B., Cusco, J.A., Roddy, M.-A., Johnstone, M.T., Creager, M.A., 1996. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J. Am. Coll. Cardiol. 27, 567–576. Zhu, L., Schwegler-Berry, D., Castranova, V., He, P., 2004. Internalization of caveolin1 scaffolding domain facilitated by antennapedia homeodomain atteneuates PAF-induced increase in microvessel permability. Am. J. Physiol. Heart Circ. Physiol. 286, H195–H201.