The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries
Objective— The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells.
Methods and Results— In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1S225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations, and Ca2+ sensitivity. The lack of function of the Sk1S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca2+ sensitivity and myogenic tone were attenuated, whereas Ca2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca2+ sensitivity) and intracellular effects of S1P (Ca2+ handling). Accordingly, S1P2 receptor inhibition (1μmol/L JTE013) attenuated myogenic tone without effect on Ca2+.
Conclusions— Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.
When transmural pressure increases, resistance arteries undergo myogenic vasoconstriction, an intrinsic response of vascular smooth muscle cells (VSMCs) to elevated mechanical load.1 Under normal physiological conditions, myogenic vasoconstriction acts as a critical element in the regulation of tissue perfusion, which also leads to further augmentation of systemic blood pressure. Several cellular events and signaling mediators have been proposed to control myogenic vasoconstriction2; we have recently identified sphingosine kinase 1 (Sk1) and its product, sphingosine-1-phosphate (S1P), as mandatory components of the pressure-induced signaling cascade.3
Although Sk1 is activated in response to elevated transmural pressure, the mechanisms through which its product, S1P, act are only partially described. S1P can activate signaling cascades as both an intracellular second messenger and as an extracellular receptor ligand (via the S1P-specific membrane receptors S1P1, S1P3 and S1P5).4 Although the intracellular targets of S1P are not well described, S1P receptor stimulation via extracellular S1P is known to activate RhoA/Rho kinase3,5 and stimulate the generation of reactive oxygen species,6 purportedly via the small GTPase Rac. Both of these signaling events possess a modulatory role in the control of the Ca2+ sensitivity of the VSMC contractile apparatus, with important functional consequences with respect to myogenic vasoconstriction.3,6
In the absence of stimulation, Sk1 possesses basal catalytic activity. Several stimuli (eg, TNF-α, growth factors, cellular depolarization) have been shown to increase Sk1 activity, which appears to result from ERK1/2 kinase-mediated phosphorylation of Sk1 at serine 225 (Ser225).7 In addition to increasing catalytic activity, phosphorylation of Ser225 also stimulates the translocation of Sk1 to the plasma membrane.7 This translocation to the plasma membrane is a prerequisite for the postulated release of S1P to the extracellular space, where it can then act as an autocrine/paracrine ligand for the S1P receptors. Although we have previously shown that activation of Sk1 is necessary for myogenic vasoconstriction,3 it is not clear what the significance of the alterations in its subcellular localization (and hence, potentially increased catalytic activity) is in microvascular smooth muscle cells.
In the present study, we investigated the role of Sk1 phosphorylation and translocation in the control of vascular tone, with special focus on myogenic vasoconstriction. We hypothesized: (1) that phosphorylation of Sk1 at serine 225 is necessary to allow for this translocation; (2) that the inability to phosphorylate Sk1 (ie, a S225A mutation) would prevent its translocation and microvascular function, rendering it with a phenotype similar to a catalytically inactive enzyme; and (3) that forced localization of Sk1 to the plasma membrane (via a myristoylation/palmitylation motif) would rescue the deficits caused by the inability to phosphorylate/translocate Sk1.
Materials and Methods
Functional and Molecular Studies Using Resistance Arteries
All animal care and experimental protocols were conducted in accordance with German and Canadian federal animal protection laws. For functional studies, which included genetic modification of resistance arteries, we used our previously described experimental model of hamster resistance arteries8 (supplement materials, available online at http://atvb.ahajournals.org). The resistance vessels used for functional studies possessed an average maximal diameter of 227±4 μm (n=65) at 45 mm Hg TMP. All experiments were conducted at a TMP of 45 mm Hg, with the exception of pressure-induced responses, which were induced by a rapid step-change from 45 mm Hg to 110 mm Hg. Before functional experiments, resistance artery smooth muscle cells were loaded with Fura 2-AM (a standard Ca2+-sensitive indicator). Resistance artery smooth muscle cell intracellular Ca2+ and outer vessel diameter were measured simultaneously, as previously described.9 Vessel viability at the start of experiments was assessed by initiating vasomotor responses with 0.3 μmol/L noradrenaline followed by 1 μmol/L acetylcholine. Vessels that did not robustly constrict to noradrenaline (>30%) or fully dilate to acetylcholine were deemed to be damaged and excluded.
To monitor Erk1/2 phosphorylation and Sk1-GFP translocation, cannulated mouse mesenteric arteries and cultured smooth muscle cells derived from these arteries were used (supplement materials).
Calculation of Tone, Reversal of Initial Distension (Myogenic Responses), and Apparent Ca2+ Sensitivity
All values of tone represent acute diameter measurements that have been normalized. The values represent the magnitude of vessel constriction relative to maximal diameter (measured using 0 mmol/L Ca2+ under depolarizing (120 mmol/L K+) conditions). The computation of tone is as follows: tone (% of diamax)=(diamax− diameasured)/diamax×100.
Myogenic vasoconstriction was initiated by a stepwise change in transmural pressure from 45 mm Hg to 110 mm Hg. The immediate passive distension of the vessel from its resting diameter is reversed by a continuous, active vasoconstriction. The “reversal of initial distension” (ie, the magnitude of the myogenic response) is calculated as the % constriction compared to the initial distension, and is computed as: reversal of initial distension (%)=(diadist−diat=7)/(diadist−diat=0)×100%, where diat=0 is the diameter immediately preceding the pressure step, diadist is the distended diameter measured immediately after the pressure step, and diat=7 is the diameter measured 7 minutes after the pressure step, an arbitrary time point where the constriction is normally stable.
The apparent Ca2+ sensitivity of the contractile apparatus was assessed as the relationship between microvascular tone and intracellular Ca2+ levels. Intracellular Ca2+ levels were adjusted by increasing extracellular Ca2+ (Ca2+ex) from 0 to 3 mmol/L under depolarizing conditions (120 mmol/L K+).
Data represent means±SEM for n experiments. For experiments comparing vascular function in arteries transfected with GFP or Sk1S225A, an unpaired Student t test was used. Experiments involving PD98059 or JTE013 used a paired Student t test to compare pre- and postinhibitor treatment for each experimental condition separately (control or Sk1wt). For analysis of multiple groups, an analysis of variance (ANOVA) followed by a t test with Bonferroni correction for multiple comparisons was employed. Differences were considered significant at error probabilities of P<0.05.
Erk1/2 Phosphorylation Stimulates Sk1 Translocation via Serine 225
Elevation of transmural pressure from 45 mm Hg to 110 mm Hg stimulated a rapid, 2-fold increase in Erk1/2 phosphorylation in mouse mesenteric arteries (Figure 1A). Erk1/2-mediated phosphorylation of Sk1 by is required for its translocation and full enzymatic activation.10 We demonstrate Sk1 translocation (using a GFP-labeled construct; Sk1wt-GFP) in primary mesenteric artery smooth muscle cells in response to 100 nmol/L S1P, a potent ligand-dependent activator of Erk1/2 (Figure 1B and 1C). Consistent with our expectations, the nonphosphorylatable Sk1 mutant (Sk1S225A-CFP) did not translocate in response to S1P (Figure 1C). The addition of a myristoylation/palmitylation motif to either construct (mp-Sk1wt-CFP or mp-Sk1S225A-CFP)10 forcibly targeted them to the plasma membrane in the absence of stimulation (Figure 1D). Technical challenges prevented reliable membrane localization of the GFP-labeled constructs in isolated resistance arteries (supplement materials).
Functional Effects of Erk1/2-Dependent Sk1 Phosphorylation in Isolated Arteries
Compared to resistance arteries expressing GFP, resistance arteries expressing the nonphosphorylatable Sk1S225A mutant displayed reduced resting tone and Ca2+, attenuated myogenic vasoconstriction and diminished pressure-stimulated Ca2+ elevation (Figure 2). Maximal diameters (diamax GFP: 208±13 μm, n=11; diamax Sk1S225A: 212±3 μm, n=7; P>0.05), vasoconstrictor responses to 0.3 μmol/L noradrenaline (GFP: 51±2%, n=11; Sk1S225A: 49±2% of diamax, n=7; P>0.05) and vasodilator responses to 3μmol/L acetylcholine (full dilation in both groups) were not affected by the expression of Sk1S225A.
To complement the experiments employing the Sk1S225A mutant, we used a MEK inhibitor, PD98059, to assess the effect of Erk1/2 inhibition on resting tone and myogenic vasoconstriction. In cultured, but nontransfected control arteries, PD98059 (10 μmol/L for 30 minutes) yielded similar effects as observed for Sk1S225A expression: reduced resting tone and Ca2+, attenuated myogenic vasoconstriction, and diminished pressure-stimulated Ca2+ elevation (Figure 3). A similar inhibitory profile was observed for arteries transfected with Sk1wt (ie, Sk1 overexpression; Figure 3).
In contrast to arteries expressing Sk1S225A, PD98059 significantly attenuated noradrenaline-stimulated vasoconstriction in both control (pre-PD: 50±2%, PD10 μmol/L: 31±3%; n=11, P<0.05) and Sk1wt-transfected arteries (pre-PD: 41±4%, PD10 μmol/L: 20±6; n=5; P<0.05). There was no statistical difference in terms of the degree of PD98059-mediated inhibition between the 2 groups (ie, P>0.05 comparing PD98059-treated control and Sk1wt-expressing arteries; unpaired t test). Vasodilator responses to 3 μmol/L acetylcholine were not affected by the inhibitor (full dilation observed).
S1P signaling is a prominent modulator of Ca2+ sensitivity (the degree of constriction for a given intracellular Ca2+ concentration).3 Arteries expressing Sk1S225A displayed significantly reduced Ca2+ sensitivity (a rightward shift in the tone/calcium relationship) compared to those expressing GFP (Figure 4A). In acute nontransfected control arteries, PD98059 elicited a similar reduction in Ca2+ sensitivity (Figure 4B).
Forced Localization of Sk1 Mutants to the Plasma Membrane
As shown in Figure 5A, the addition of a membrane-targeting motif to the Sk1S225A mutant (mp-Sk1S225A) only partially rescued resting tone (P<0.05 compared to both GFP and Sk1S225A from Figure 2A) and apparent Ca2+-sensitivity remained substantially reduced (Figure 5B). In addition, membrane targeting did not restore myogenic vasoconstriction (P<0.05 comparing Sk1S225A from Figure 2B and mp-Sk1S225A from Figure 5C); however, resting and pressure-stimulated Ca2+ elevation were completely rescued to control levels (Figure 5D).
We have previously shown that overexpression of Sk1wt enhances resting tone, myogenic vasoconstriction, and pressure-induced Ca2+ elevation.3 We were therefore surprised to observe that the addition of the membrane-targeting motif to the wild-type Sk1 (mp-Sk1wt) was associated with reduced resting tone, Ca2+ sensitivity, and myogenic vasoconstriction (Figure 5). Of significance, expression of mp-Sk1wt inhibited Ca2+ sensitivity and myogenic vasoconstriction to a lesser extent than mp-Sk1S225A. Resting intracellular Ca2+ levels in resistance arteries expressing mp-Sk1wt were not different from controls and the pressure-induced increase in Ca2+ resembled that seen in arteries expressing Sk1wt (ie, they had a higher amplitude than control arteries3; Figure 5D).
Vasomotor responses to 0.3 μmol/L noradrenaline (mp-Sk1wt: 48±2%, n=6; mp-Sk1S225A: 45±3%, n=6; P>0.05) and 3 μmol/L acetylcholine (maximal dilation in both groups) were not affected by the expression of either membrane-anchored Sk1 mutant.
Involvement of the S1P2 Receptor
The effects of extracellular S1P are predominantly mediated via the S1P2 receptor subtype in hamster resistance arteries.11 To assess the contribution of this receptor, we used the specific S1P2 inhibitor, JTE013. We have previously documented that 1 μmol/L JTE013 is sufficient to inhibit vasoconstrictor responses to applied S1P.12 In cultured, but nontransfected control arteries, 1μmol/L JTE013 (30 minutes) attenuated myogenic vasoconstriction, but did not affect resting tone, resting Ca2+ or the pressure-induced elevation of Ca2+ (Figure 6). In arteries overexpressing Sk1wt, JTE013 reduced both resting tone and myogenic vasoconstriction, without significant effect on resting Ca2+ or the pressure-induced elevation of Ca2+ (Figure 6). The magnitude of myogenic vasoconstriction observed in both JTE013-treated groups was similar (both ≈75% reversal of initial distension).
Sphingosine-1-phosphate (S1P) is a critical determinant of resting and myogenic tone in resistance arteries.3 As such, S1P levels must be tightly regulated to maintain the appropriate level of constriction in resistance arteries. Failure to adequately control artery resistance would negatively impact capillary perfusion and could affect systemic blood pressure.
In this regard, the synthesis of S1P appears to be effectively limited under nonstimulated conditions through the spatial separation of the sphingosine kinase 1 (Sk1) enzyme from its substrate, sphingosine. In this resting state, Sk1 possesses constitutive catalytic activity,10 but is primarily localized to the cytosolic compartment (ie, away from its substrate). The spatial inhibition is overcome when activated Sk1 translocates to the plasma membrane, where it then provides S1P to both the intra- and extracellular compartments.
The two pools of S1P have distinct signaling properties in vascular smooth muscle cells: extracellular S1P activates cell surface receptors (predominantly the S1P2 receptor in hamster resistance arteries11), which primarily initiate small GTPase signaling cascades,3,4,6,13 whereas intracellular S1P stimulates the release of Ca2+ from intracellular stores.14–16 Because we postulate that both S1P pools play significant roles in the regulation of microvascular tone,3,4,6,13 plasma membrane localization of activated Sk1 would be an ideal position to supply both compartments with S1P.
Our previous investigation in isolated arteries demonstrated Sk1wt-GFP translocation as an indication of pressure-dependent activation, without elucidating the underlying mechanism.6 In this regard, Erk1/2 phosphorylation is deemed to be the critical initiating step for Sk1 translocation/activation.7,10 A key observation of the present study, therefore, was that elevation of transmural pressure stimulated rapid (ie, within 15 seconds) Erk1/2 phosphorylation (Figure 1A). In support of the critical role of Erk1/2 phosphorylation, Sk1 translocation was absent in cultured smooth muscle cells expressing a GFP-coupled Sk1 mutant lacking the Erk1/2 phosphorylation site (Sk1S225A-GFP).
The functional consequences of expressing Sk1S225A in isolated resistance arteries were consistent with a lack of Sk1 activation. Specifically, Sk1S225A expression reduced resting and stimulated intracellular Ca2+ levels, resting tone, Ca2+ sensitivity and myogenic vasoconstriction.
These observations are particularly interesting because the Sk1S255A mutant retains basal catalytic activity.10 Therefore, the primary factor that renders it with a phenotype similar to a Sk1 mutant without catalytic activity (Sk1G82D)3 is its inability to be phosphorylated/translocated.
Experiments using activated macrophages and neuroblastoma cells have revealed a second phosphorylation-independent translocation mechanism that is sensitive to Ca2+/calmodulin inhibition.17,18 Any Ca2+/calmodulin-based mechanism could conceivably be activated by the pressure-induced Ca2+ elevation in our system. Our data on tone regulation, however, do not support that phosphorylation-independent translocation of Sk1 contributes to any great extent in vascular smooth muscle cells. Taken together, the evidence guides two important conclusions: (1) in resistance artery vascular smooth muscle cells, Sk1 translocation appears to be primarily dependent on phosphorylation, and (2) translocation of Sk1 is required for its regulatory function with respect to myogenic vasoconstriction.
To support our conclusions regarding the involvement of ERK1/2 in Sk1 phosphorylation/activation, we inhibited MEK with PD98059, expecting that a similar phenotype as with the Sk1S225A mutant would result. Indeed, MEK inhibition, in both control and Sk1wt-transfected arteries, resulted in a functional profile that was remarkably similar to that of Sk1S225A, including decreased resting tone and Ca2+, an attenuated myogenic vasoconstriction and reduced Ca2+ sensitivity and diminished pressure-dependent Ca2+ elevation. In addition to the effects mimicking Sk1S225A expression, PD98059 also attenuated norepinephrine-stimulated vasoconstriction. Collectively, these results are in accordance with the inhibition of myogenic tone and vasomotor responses to vasopressin and KCl shown by Lagaud and coworkers.19
A likely explanation for the broader effects of PD98059 lies in the fact that Sk1S225A expression and Erk1/2 inhibition are not equivalent inhibitory strategies. Although the Sk1S225A mutant specifically interrupts signaling between Erk1/2 and Sk1, PD98059 globally blocks all Erk1/2-dependent processes. Because Erk1/2 has several downstream effectors that are independent of Sk1, we were not surprised to observe that PD98059 had additional vascular effects compared to Sk1S225A (ie, attenuation of norepinephrine responsiveness).
At this point, our data indicated that the Erk1/2-dependent phosphorylation and translocation of Sk1 is mandatory for its function. Several reasons may contribute as to why: (1) it potentially allows for localization to plasma membrane microdomains (eg, caveolae), which are enriched with its substrate, sphingosine, and offer the opportunity to cluster with other S1P-related signaling components; (2) localization to the membrane would be a necessary prerequisite for the previously described export of S1P and/or Sk120; and (3) exposure of Sk1 to alterations in electric potential, which may modulate its activity,21 can only occur at the plasma membrane. We therefore aimed to bypass the translocation deficiency of the Sk1S225A mutant by the addition of a myristoylation/palmitylation motif that acts as a membrane anchor (mp-Sk1S225A). We hypothesized that forced localization of Sk1S225A to the plasma membrane would restore its function. However, this forced localization maneuver was only partially successful. Although intracellular Ca2+ signals, both resting and stimulated, were fully restored, apparent Ca2+ sensitivity and myogenic tone, two parameters that heavily depend on receptor-mediated S1P signaling, did not benefit. These results suggest the existence of at least two “S1P signaling compartments.” Given the known dual role of S1P as both an extracellular ligand and an intracellular second messenger,4 the most straight-forward compartmentalization would be into intra- and extracellular S1P pools. In this regard, the forced localization of the Sk1S225A mutant to the plasma membrane (mp-Sk1S225A) was sufficient to rescue effects attributed to intracellular S1P, but it clearly failed to restore the effects mediated by extracellular S1P. These differential effects may result from incorrect subcellular positioning of Sk1 by the fused membrane anchor, which could then limit the ability of Sk1 to supply S1P to the extracellular compartment. This interpretation would be consistent with observations made in a fundamentally different experimental system of transfected 3T3-L1 fibroblasts.22
If our interpretation is correct, then the interruption of cell surface receptor-mediated S1P signaling should yield a similar profile as mp-Sk1S225A. To address this hypothesis, we used the selective S1P2 receptor antagonist, JTE013. Similar to mp-Sk1S225A, JTE013 attenuated myogenic vasoconstriction in both control (cultured, but nontransfected) and Sk1wt-expressing vessels, without effect on resting or pressure-induced Ca2+ levels.
Combined, our results (1) confirm our initial hypothesis that Sk1 must translocate to the plasma membrane in order to exert its full spectrum of effects, and (2) indicate that Sk1 is precisely targeted by the translocation mechanism. Simply forcing an association between the Sk1 enzyme and plasma membrane does not confer all of the functional effects that the endogenous translocation process does.
We therefore hypothesized that the efficacy of the wild-type enzyme would also be severely impaired by the addition of the same membrane anchor. We have previously documented that overexpression of Sk1wt substantially enhances pressure-induced Ca2+ levels and myogenic vasoconstriction.3 Consistent with our hypothesis, the fused membrane anchor inhibited the normal function of Sk1. Specifically, myogenic vasoconstriction was substantially attenuated. Remarkably, pressure-induced Ca2+ levels were virtually identical to those observed after Sk1wt overexpression3 (ie, significantly higher than in the respective controls, which were GFP-expressing arteries). Again, the most straightforward explanation for these observations would be the separation of these two functions into two different compartments (ie, cell surface receptor effects on Ca2+ sensitivity and intracellular effects on Ca2+ handling).
We observed that the mp-Sk1wt construct enhanced pressure-induced Ca2+ elevation, whereas expression of mp-Sk1S225A did not, suggesting that serine 225 phosphorylation does more than simply initiate the translocation of Sk1. Because catalytic activity is enhanced by phosphorylation of serine 225,7,10 this likely underlies the difference between the two constructs. Thus, resistance arteries expressing the mp-Sk1wt may generate more S1P under basal and stimulated conditions than those expressing mp-Sk1S225A.
Ca2+ sensitivity and myogenic tone, two effects reliant on extracellular S1P, were also stronger in vessels expressing mp-Sk1wt compared to those expressing mp-Sk1S225A. We therefore speculate that at least a small proportion of the intracellularly generated S1P could have been released to the extracellular compartment, resulting in S1P receptor activation. Although our experimental system is not conducive for the measurement of S1P concentrations in either compartment, release of S1P from cells expressing the membrane anchored10 and nonphosphorylatable (Sk1S225A) Sk1 mutant is conceivable and has been demonstrated.7
In summary, we show that phosphorylation-dependent activation and translocation of Sk1 is critical for its regulatory function in vascular smooth muscle cells. This complex process cannot be imitated by an artificial membrane anchor, indicating that the association of Sk1 with the plasma membrane is a precisely targeted process. The present study highlights the significance of subcellular localization of Sk1 as a governing mechanism for the control of smooth muscle function. However, to fully appreciate the control of Sk1 localization, future work needs to elucidate the mechanisms that direct the translocation process (ie, the events that lie between Sk1 phosphorylation and correct plasma membrane integration). Such advancement in our knowledge could lead to the development of highly effective therapeutical tools that modulate smooth muscle function for clinical use in the manipulation of vessel tone regulation.
The authors thank Aileen Roman, Sabine D’Avis, Marold Buchner, and Franz Singer for their technical assistance with data collection.
Sources of Funding
This work was supported by a Canadian Institutes of Health Research Fellowship (DL, FRN#63761), European Union Exgenesis Grant LSHM-CT-2004-005272 (U.P.) start-up funding from the University of Toronto (to S.-S.B.), a New Investigator Award and research grant from the Heart and Stroke Foundation of Ontario (to S.-S.B., NA6198), and a joint infrastructure grant from the Canadian Foundation for Innovation and Ontario Research Fund (to S.-S.B., 11810).
Received March 16, 2009; revision accepted August 18, 2009.
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