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Am J Physiol Heart Circ Physiol 275: H1898-H1904, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 5, H1898-H1904, November 1998

RAPID COMMUNICATION
Phosphatidylinositol 3-kinase gamma  mediates shear stress-dependent activation of JNK in endothelial cells

Young-Mi Go1, Heonyong Park1, Matthew C. Maland1, Victor M. Darley-Usmar1, Borislav Stoyanov2, Reinhard Wetzker2, and Hanjoong Jo1

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2 Max Planck Research Unit Molecular Cell Biology, University of Jena, 07747 Jena, Germany

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Shear stress differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) by mechanisms involving Galpha i2 and Gbeta /gamma proteins, respectively, in bovine aortic endothelial cells (BAEC). The early events in this signaling mechanism by which G proteins regulate ERK and JNK in response to shear stress have not been defined. Here we show that BAEC endogenously express a G protein-dependent form of phosphatidylinositol 3-kinase, PI3Kgamma , and its activity is stimulated by shear stress. PI3Kgamma activity was measured in vitro using BAEC that were transiently transfected with an epitope-tagged PI3Kgamma (vsv-PI3Kgamma ). Exposure of BAEC to shear stress rapidly and transiently stimulated the activity of vsv-PI3Kgamma (maximum by 15 s, with a return to basal after 1-min exposure to 5 dyn/cm2 shear stress). Activity of vsv-PI3Kgamma was stimulated by shear stress intensities as low as 0.5 dyn/cm2. Treatment of BAEC with an inhibitor of PI3K, wortmannin, inhibited shear-dependent activation of JNK but had no effect on that of ERK. Furthermore, expression of a kinase-inactive mutant (PI3Kgamma K799R) in BAEC inhibited the shear-dependent activation of JNK but not ERK. Taken together, these results suggest that PI3Kgamma selectively regulates the shear-sensitive JNK pathway. This differential and novel signaling pathway may be responsible for coordinating various mechanosensitive events in endothelial cells.

mechanotransduction; extracellular signal-regulated kinase; G proteins; atherosclerosis

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

VASCULAR ENDOTHELIAL CELLS are constantly exposed to hemodynamic shear stress, the dragging force generated by blood flow. Shear stress controls vascular tone, vessel wall remodeling, the interaction of blood cells with endothelium, coagulation, and fibrinolysis (4). The focal pattern of atherosclerotic lesions in areas of low and/or unstable shear stress further highlights the importance of this physical force in the atherogenic process (15, 44). Endothelial cells play a key role in shear-dependent vascular changes, sensing shear stress by an unidentified mechanoreceptor(s) that leads to the production of autocrine and paracrine factors (4). For example, shear stress regulates expression of many genes, including adhesion molecules, growth factors, superoxide dismutases, endothelial nitric oxide synthase, endothelin, monocyte chemoattractant protein-1 (MCP-1), tissue factors, and others, at the level of gene transcription (6, 20, 26-33, 35, 40, 41). A conserved shear stress-response element has been identified initially in the 5'-promoter region of platelet-derived growth factor B gene and subsequently in many other mechanosensitive genes (14, 33). Another shear-sensitive cis-acting element, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate-responsive element, has been found in the 5'-promoter sequence of the MCP-1 gene (35). Furthermore, transcription factors (nuclear factor-kappa B and activator protein 1) and immediate-early response genes (c-fos, c-jun, and c-myc) have been shown to be regulated by shear stress (10, 17). In addition, many of these nuclear responses have been shown to be controlled, in large part, by mitogen-activated protein (MAP) kinases (3, 13, 19).

Shear stress has been shown to stimulate two members of the MAP kinase family, extracellular signal- regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK; also called stress-activated protein kinase) (12, 18, 19, 42, 43). Furthermore, we have shown that shear stress differentially regulates activation of ERK and JNK by mechanisms involving Galpha i2 and Gbeta /gamma , respectively, in bovine aortic endothelial cells (BAEC) (12). However, the signaling components and the sequence of signaling events that link Galpha i2 and Gbeta /gamma to activation of ERK and JNK, respectively, have not been determined.

Recently, a new form of phosphatidylinositol 3-kinase, PI3Kgamma , which is regulated by alpha - and beta /gamma -subunits of heterotrimeric G proteins, has been cloned from cDNA libraries of human U-937 cells and pig neutrophils (36, 37). This growing family of PI3K now includes PI3Kalpha /beta , PI3Kgamma , PI3K-68D, and VPS34p forms (2, 36). PI3Kalpha and PI3Kbeta are heterodimers, which are composed of p110 catalytic and p85 regulatory subunits, and the enzymatic activity of the p110 subunit is controlled by binding of the p85 subunit (2). Typically, PI3Kalpha and PI3Kbeta are activated by mechanisms involving receptor tyrosine kinases (2). In contrast, the catalytic subunit of PI3Kgamma (p110gamma ) does not bind to the p85 regulatory subunit (36, 37). Instead, the activity of p110gamma has been shown to be controlled by Galpha and Gbeta /gamma proteins that are believed to bind to a pleckstrin homology domain found in the NH2-terminal region of PI3Kgamma (36, 37). Furthermore, it has been shown that PI3Kgamma mediates activation of ERK and JNK in response to Gbeta /gamma or agonists acting on G protein-coupled receptors (23, 24).

Because G protein-dependent events are critical in the mechanosensitive activation of both ERK and JNK, we examined whether the G protein-sensitive PI3Kgamma mediates these signal transduction pathways. In the present study, the effect of shear stress on PI3Kgamma activity was characterized in BAEC. To examine the role of PI3Kgamma , shear stress-dependent activation of ERK and JNK was studied using BAEC, in which PI3Kgamma activity was inhibited by either treatment with wortmannin or transient expression of a kinase-inactive mutant, PI3Kgamma K799R.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. BAEC harvested from descending thoracic aortas were maintained (37°C, 5% CO2) in a growth medium [DMEM (1 g/l glucose; GIBCO) containing 20% FCS (Atlanta Biologicals) without antibiotics] (12). Cells used in this study were between passages 5 and 10. For shear stress experiments, 1 × 106 cells per glass slide (75 × 38 mm, Fisher Scientific) were seeded in the growth medium. The next day, the medium was changed to a starvation medium (phenol red-free DMEM containing 0.5% FCS and 25 mM HEPES) and incubated for 16 h.

Plasmids, adenovirus, and transfection. Plasmids encoding for hemagglutinin (HA)-tagged ERK2 (HA-ERK2), HA-JNK1, and c-Jun (amino acids 5-89) fused to glutathione S-transferase (GST-c-Jun) were described previously (12). To aid in the immune precipitation of PI3Kgamma for the in vitro lipid kinase assay, the 11-amino acid epitope sequence (YTDIEMNRLGK) of vesicular stomatitis virus (vsv) glycoprotein was inserted into the unique Sac I site found at codon 36 of the coding sequence for PI3Kgamma (37). Two complementary synthetic primers (30 pmol) coding for the epitope sequence and additional Sac I sites were hybridized to each other and ligated into the Sac I-digested pBluescript vector containing the PI3Kgamma coding sequence (37) to generate the vsv-PI3Kgamma construct. The junction site was verified by DNA sequencing of the construct. The tagged construct was then subcloned into a pcDNA3 mammalian expression vector using BamH I/Xho I sites. A kinase-inactive mutant, PI3Kgamma K799R, which was prepared by a point mutation of the DNA sequence coding for Lys799 to Arg799, has been described previously (23, 24). Endotoxin-free plasmids used in all transfection experiments were prepared by using a maxiprep kit (Quiagen) and following the manufacturer's instructions.

For transfection studies, BAEC (2.5 × 105 cells/glass plate) were grown overnight in the growth medium and transfected by a method using the adenovirus conjugated to polylysine (AdpL) as described previously (12). Under identical conditions, this method yielded ~25-35% transfection efficiency of beta -galactosidase DNA in pCMV (cytomegalovirus) vector (American Type Culture Collection) as determined by a histochemical staining method using 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (12).

Shear stress and preparation of lysates. The glass slide containing a BAEC monolayer was assembled into a parallel-plate shear chamber, forming a flow channel (220 µm high × 2.5 cm wide × 6.2 cm long) between the monolayer and fabricated polycarbonate plate as described previously (16). Nonpulsatile, laminar shear stress was controlled by changing the flow rate of the starvation medium delivered to the cells using the constant-head flow loop or a syringe pump (KD Scientific) as described previously (11, 16).

After treatment, BAEC were washed in ice-cold PBS and lysed in 0.25 ml of lysis buffer A [10 mM beta -glycerophosphate, pH 7.6, 20 mM HEPES, 20 mM MgCl2, 20 mM p-nitrophenyl phosphate, 0.1 mM vanadate, 2 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100] for ERK and JNK assays or lysis buffer B (20 mM HEPES, pH 7.6, 2 mM EGTA, 0.2 mM EDTA, 5 mM beta -glycerophosphate, 1 mM vanadate, 3 mM MgCl2, 120 mM NaCl, 10 µg/ml leupeptin, 2 µg/ml pepstatin, 1 mM benzamidine, 0.1 mM PMSF, and 1% Nonidet P-40) for PI3K assay. Cell lysates were clarified by spinning at 20,000 g for 15 min at 4°C. Protein content of each sample was measured by using a Bio-Rad DC kit.

Western blot analysis of PI3Kgamma . To detect PI3Kgamma , aliquots of lysates were resolved on 7.5% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), and probed with a mouse monoclonal PI3Kgamma antibody (23). Goat anti-mouse IgG conjugated to alkaline phosphatase was used as secondary antibody and developed by a chemiluminescent detection method (12).

MAP kinase assays. ERK activation in cell lysates (10 µg) was examined by Western blot analysis using an antibody specific to an active, phosphorylated form of ERK (phospho-ERK; New England Biolabs) as described previously (12). As a control, the total amount of ERK was determined by using a p44/42 MAPK antibody (New England Biolabs; data not shown).

For immune complex assays, antibodies specific for JNK1 (clone no. G151-333, Pharmingen) and HA (Boehringer Mannheim) were incubated with the soluble lysates (100 µg) for 1 h at 4°C, followed by an additional 1-h incubation with protein A- (for HA antibodies) or protein G-agarose (for JNK1 antibody) beads. The immune complex was washed four times in lysis buffer A and twice in buffer C (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM beta -glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM vanadate, and 2 mM DTT). The washed immune complexes were incubated in 20 µl of buffer C containing either myelin basic protein or GST-c-Jun (5 µg each) and 5 µCi of [gamma -32P]ATP for 20 min at 30°C. The reaction products were resolved by SDS-PAGE and transferred to a PVDF membrane, an autoradiogram was obtained, and the radioactivity incorporated into each band was quantified by scintillation counting. The membrane was then probed with antibodies to p44/42 MAPK or JNK (9) to monitor the total amount of immunoprecipitated ERK and JNK in each experiment.

In vitro PI3K assay. Soluble lysates obtained from BAEC transfected with vsv-PI3Kgamma were incubated with a vsv antibody (Sigma) for 1 h, followed by incubation with protein G-agarose for another hour. The immune complex was washed in lysis buffer B four times and once in a kinase buffer (40 mM HEPES, pH 7.4, 2 mM EGTA, 0.2 mM EDTA, 1 mM DTT, 100 mM NaCl, 1 mM beta -glycerophosphate, 0.1 mM vanadate, and 4 mM MgCl2). The specific immunoprecipitation of vsv-PI3Kgamma was confirmed by Western blot analysis using a monoclonal PI3Kgamma antibody (data not shown). In vitro PI3Kgamma assay was performed as previously described (37). Briefly, phosphatidylinositol (PI; 30 µg/sample) was dried under N2 gas, resuspended in the kinase buffer, and sonicated four times for 15 s each. The PI vesicles (30 µl) were added to the immune complex, and then this mixture was incubated for 10 min on ice. Phosphorylation of PI was initiated by adding 20 µl of a reaction buffer (20 mM MgCl2, 50 µM ATP, and 10 µCi [gamma -32P]ATP) to the above mixture at 30°C for 5 min. The reaction was terminated by adding 15 µl of 4 N HCl and mixing in MeOH:CHCl3 (1:1). PI-phosphate (PI-P) was analyzed by TLC, and autoradiograms were obtained as described previously (37). Radioactivity incorporated into a PI-P spot was quantified by scintillation counting.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of endogenous and transfected PI3Kgamma in BAEC. PI3Kgamma has been shown to be expressed in the human leukemic cell lines (K-562 and U-937 cells) and pig neutrophils (36, 37). To determine whether endothelial cells also express the enzyme, cell lysates obtained from BAEC were analyzed by Western blot using a monoclonal PI3Kgamma antibody. As expected, the monoclonal antibody raised against human PI3Kgamma recognized a band with a molecular mass of 110 kDa in the K-562 cell lysate (Fig. 1, lane 1). The antibody also reacted with a p110 band in BAEC (Fig. 1, lane 2). However, the staining intensity of the p110 band in BAEC lysate (Fig. 1, lane 2 loaded with 80 µg protein) was significantly lower than that of K-562 cells (Fig. 1, lane 1 loaded with 25 µg protein). This result is caused by either the relatively low expression level of PI3Kgamma or the weak cross-reactivity of the bovine enzyme against the monoclonal antibody raised against the human counterpart. In BAEC, PI3Kgamma was only found in the Triton-soluble lysate, not in the insoluble fraction (data not shown). To determine whether shear stress regulates activity of PI3Kgamma , we decided to tag the enzyme with an epitope (vsv) so that the functional enzyme could be immunoprecipitated and used for a subsequent in vitro kinase assay. vsv-PI3Kgamma fusion protein (~p120) can be transiently expressed in BAEC as determined by Western blot analysis (Fig. 1, lane 3).


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  		Fig. 1.   Detection of endogenous and recombinantly expressed phosphatidylinositol 3-kinase gamma  (PIK3gamma ) in bovine aortic endothelial cells (BAEC). Cell lysates obtained from K-562 cells (25 µg; lane 1), BAEC (80 µg; lane 2,) and BAEC transiently transfected with vesicular stomatitis virus (vsv)-PI3Kgamma (20 µg; lane 3) were analyzed by Western blot using a PI3Kgamma antibody. For transfection studies, BAEC were transfected with cDNA encoding vsv-PI3Kgamma by a method using adenovirus conjugated to polylysine (AdpL). Two days after transfection, cell lysates were prepared. The 110-kDa bands (lanes 1 and 2) and a band migrating slightly lower than the p110 because of vsv epitope (lane 3), which correspond to the molecular masses of PI3Kgamma and vsv-PI3Kgamma , respectively, are indicated by arrowhead.

Shear stress stimulates PI3Kgamma activity. We next determined the role of shear stress on the transiently expressed vsv-PI3Kgamma fusion protein. vsv-PI3Kgamma was immunoprecipitated from BAEC lysates by using a vsv antibody and protein G-agarose, and the immune complex was used to determine the lipid kinase activity in vitro. Shear stress rapidly and transiently stimulated vsv-PI3Kgamma activity, reaching a maximum by 15 s and returning to a basal level by 1 min (Fig. 2B). The activity of vsv-PI3Kgamma was maximally stimulated at all shear magnitudes tested in the present study (0.5 dyn/cm2 and higher) (Fig. 2A). To further confirm the specificity of PI3Kgamma activity, BAEC were incubated with a PI3K inhibitor, wortmannin. Shear stress-dependent activation of PI3Kgamma was inhibited by pretreating BAEC with 100 nM wortmannin for 10 min (Fig. 2A). This result is consistent with previous findings (36, 37, 39), which have shown that PI3Kgamma is a wortmannin-sensitive, G protein-dependent PI3K.


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  		Fig. 2.   Shear stress stimulates PI3Kgamma activity in a time-dependent manner. Two days after transfection with vsv-PI3Kgamma in BAEC, cells were subjected to increasing force levels of shear stress for 15 s (A) or to 5 dyn/cm2 shear stress for time periods indicated (B). In some experiments, cells were pretreated with 100 nM wortmannin for 10 min (A, open circle ) before shear exposure (5 dyn/cm2 for 15 s). vsv-PI3Kgamma was immunoprecipitated from cell lysates by using a vsv antibody, and the immune complex was used to phosphorylate phosphatidylinositol (PI) in presence of [gamma -32P]ATP. Reaction products were separated by TLC and autoradiograms (top) were obtained. Radioactivity incorporated into phosphorylated PI spot (PI-P) in TLC plates as identified from autoradiograms was determined by scintillation counting and plotted (bottom). Values are means ± SE (n = 2-5).

PI3Kgamma mediates shear stress-dependent activation of JNK but not ERK. Previously, we showed that shear stress stimulates ERK and JNK by mechanisms involving alpha - and beta /gamma -subunits, respectively, of G proteins (12). Because shear stress stimulates the G protein-sensitive PI3Kgamma , as shown in Fig. 2, we determined whether PI3Kgamma mediates shear stress-dependent activation of ERK and JNK by using two independent approaches. First, BAEC were pretreated with wortmannin before cells were subjected to shear stress. Pretreatment of BAEC for 10 min with 100 nM wortmannin had no significant effect on shear stress-dependent activation of ERK (Fig. 3A). In contrast, pretreatment of BAEC with 100 nM wortmannin inhibited shear stress-dependent activation of JNK by ~60% compared with control (P < 0.005, n = 4) (Fig. 3B). The inhibitory effect of wortmannin on JNK activity was not caused by a decreased amount of JNK contained in the immune complex, as demonstrated by Western blot analysis using a JNK antibody (Fig. 3B, top).


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  		Fig. 3.   Wortmannin inhibits shear-dependent activation of c-Jun NH2-terminal kinase (JNK) but not extracellular signal-regulated kinase (ERK). BAEC were pretreated for 10 min in absence or presence of wortmannin (0.1 µM) before cells were subjected to 5 dyn/cm2 shear stress for 5 min (A) or 1 h (B). A: cell lysates were analyzed by Western blot with a phosphorylated ERK (pERK) antibody (top). Densitometry was performed to quantitate pERK1 and pERK2 band intensities, and activation was expressed as a percentage of maximum activation (bottom). Quantitation of pERK1 and pERK2 showed essentially identical results. Values in bar graph are means ± SE (n = 4). B: cell lysates were incubated with a JNK1 antibody, and immune complex was used to phosphorylate glutathione S-transferase (GST)-c-Jun. Reaction products were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, and an autoradiogram showing phosphorylation of GST-c-Jun (GST-c-Jun~P) was obtained. The membrane was then probed with an antibody to JNK (see Ref. 9) to examine amount of immunoprecipitated JNK in each lane (top). Radioactivity incorporated into GST-c-Jun bands was determined by scintillation counting (bottom). Values in bar graph are means ± SE (n = 4).

Because wortmannin inhibits not only PI3Kgamma but also PI3Kalpha /beta types as well, we used a kinase-inactive PI3Kgamma mutant (PI3Kgamma K799R) to determine the role of PI3Kgamma on the ERK and JNK pathways. For this study BAEC were cotransfected with HA-ERK (Fig. 4A) or HA-JNK (Fig. 4B) along with PI3Kgamma K799R or an empty vector control (pcDNA). Exposure to shear stress for 5 min increased the activity of HA-ERK (Fig. 4A, lanes 1 and 2) as shown previously (12). Coexpression of PI3Kgamma K799R did not affect the shear stress-dependent activation of HA-ERK (Fig. 4A, lanes 3 and 4). Although there was a small decrease in basal HA-ERK activity in cells expressing PI3Kgamma K799R, it was not statistically significant (P > 0.05, n = 3) (Fig. 4A, compare lane 1 with lane 3). On the other hand, coexpression of PI3Kgamma K799R completely prevented activation of HA-JNK induced by exposure to shear stress for 1 h (Fig. 4B, compare lanes 7 and 8 with lanes 5 and 6). Taken together, these results suggest that PI3Kgamma is an upstream mediator of shear stress-dependent activation of JNK but not ERK.


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  		Fig. 4.   Expression of a kinase-inactive mutant (PI3Kgamma K799) inhibits shear stress-dependent activation of JNK but not ERK. BAEC were transiently cotransfected with hemagglutinin-tagged ERK2 (HA-ERK2; A) or HA-JNK1 (B) along with PI3Kgamma K799 or an empty vector control (pcDNA) by using an AdpL method. Two days after transfection, cells were subjected to static control (0 dyn/cm2) or shear stress [5 dyn/cm2 for 5 min (A) or 1 h (B)], and cell lysates were prepared. HA-ERK2 and HA-JNK1 were immunoprecipitated from cell lysates by using an anti-HA antibody, and immune complexes were used to phosphorylate myelin basic protein (MBP; A) and GST-c-Jun (B), respectively. Reaction products were separated by SDS-PAGE and transferred to a PVDF membrane, and autoradiograms showing phosphorylation of MBP (MBP~P; A) and GST-c-Jun (GST-c-Jun~P; B) were obtained. Membranes were then probed with antibodies to ERK1/2 and JNK (see Ref. 9) to examine amount of immunoprecipitated HA-ERK2 and HA-JNK1 in each lane (top). Radioactivity incorporated into MBP and GST-c-Jun bands was determined by scintillation counting (bottom). Values in bar graphs are means ± SE (n = 3 for A; n = 4 for B).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Shear stress regulates vascular wall function and structure by mechanisms including expression of multiple genes in endothelial cells (4). Although it is likely that these events are controlled by various signaling pathways, MAP kinases including ERK and JNK have been shown (3, 13) to play key roles in linking external signals to nuclear responses including gene expression. Previous evidence (12) suggests that ERK and JNK are activated through different signal transduction pathways. Shear stress stimulates ERK rapidly and transiently (maximum by 5-min shear exposure) in a shear force-dependent manner, whereas JNK activation occurs over a much slower time course (maximum by 1-h shear exposure) and shows a maximal response at all shear forces tested (0.5 dyn/cm2 or higher) (12). It was also shown (12) that the signaling pathways regulating the shear stress-dependent activation of ERK involve Galpha i2, tyrosine kinase(s), and Ras-dependent mechanisms, whereas that of JNK requires non-pertussis toxin-sensitive Galpha , Gbeta /gamma , tyrosine kinase(s), and Ras-dependent mechanisms. More recent studies (18, 42) further demonstrated the roles of protein kinase C-epsilon and focal adhesion kinase in the mechanosensitive ERK pathway. However, the exact sequences of signaling events commencing with shear exposure and proceeding to activation of G proteins and, subsequently, MAP kinases have not been determined.

The current study demonstrates that shear stress stimulates activity of a G protein-sensitive form of PI3K, PI3Kgamma , over a relatively short and transient time course (Fig. 2). Unlike the time dependency, however, all shear levels tested in this study as low as 0.5 dyn/cm2 lead to maximal activation of PI3Kgamma , showing no force dependency (Fig. 2A). These results suggest that activation of PI3Kgamma may be dependent on initiation of flow or step change in shear force. Whether there is a minimum or threshold level of change in shear magnitude that is required to activate PI3Kgamma is still an open question. It is interesting to note, however, that activation of JNK in response to shear stress displays the same mechanical sensitivity (12) as that of PI3Kgamma reported in the current study. In contrast, shear stress-dependent activation of ERK, which is not regulated by PI3Kgamma (Figs. 3 and 4), requires a much higher level of shear force (10 dyn/cm2) for its maximal activation (12). Although the mechanisms underlying these differential mechanical sensitivities displayed by ERK and JNK pathways are not known currently, selective engagement of upstream signaling molecules such as PI3Kgamma is likely to be critical.

Activity of PI3Kgamma has been demonstrated to be dependent on Gbeta /gamma in various cell types as well as in vitro (23, 24, 36, 37, 39). Moreover, PI3Kgamma has been shown to activate JNK in a Gbeta /gamma -dependent manner in COS cells (24). Previously, we also showed (12) that JNK is activated in BAEC in a Gbeta /gamma -dependent manner. These previous reports and our current study demonstrating that PI3Kgamma is a critical upstream regulator of shear stress-dependent activation of JNK are consistent with the notion that PI3Kgamma activation is beta /gamma -subunit dependent.

We also demonstrate that wortmannin partially inhibits shear stress-dependent activation of JNK (Fig. 3). The lack of total inhibition may be caused by the presence of wortmannin-insensitive signaling pathways that are also important in regulation of shear activation of JNK. Consistent with this idea, Li et al. (18) suggested that stimulation of JNK by shear stress requires activation of both focal adhesion kinase-dependent and -independent mechanisms (18). Other possibilities include a limited availability of wortmannin in the cell signaling system because of the metabolic conversion of the inhibitor or competition of the enzyme substrates including ATP and phosphoinositides against wortmannin (38). Because wortmannin inhibits not only PI3Kgamma but also PI3Kalpha and PI3Kbeta , we could not exclude the possibility that the partial effect of wortmannin could be attributed to PI3Kalpha and PI3Kbeta . However, the selective effect of PI3Kgamma K799R (Fig. 4) indicates that PI3Kgamma plays a critical role in shear stress-dependent activation of JNK.

The effects of PI3Kgamma K799R were more pronounced than those induced by the pharmacological inhibitor of PI3K, wortmannin. The precise reasons for this discrepancy are not clear at present. PI3Kgamma K799R may act as a dominant-negative inhibitor, which may exert prolonged and potentially irreversible effects on other signaling molecules. Consistent with this possibility, PI3Kgamma K799R has been shown to prevent activation of JNK induced by Gbeta /gamma in COS-7 cells (24). In any event, both the pharmacological and molecular biological approaches consistently indicate a major role for PI3Kgamma in the shear stress-dependent activation of JNK.

In contrast to JNK, wortmannin and PI3Kgamma K799R did not have any significant effect on shear activation of ERK, revealing the signaling specificity of PI3Kgamma in endothelial cells. One of the potential mechanisms controlling this specificity may be at the level of Ras. For example, although both PI3Kalpha (p85/p110 heterodimeric form) and PI3Kgamma can bind to the GTP-bound form of Ras (Ras-GTP), GTP-Ras stimulated only the activity of PI3Kalpha , not PI3Kgamma (34). This differential regulatory mechanism could be mediated by adapter molecules such as the p85 regulatory subunit, which is present in PI3Kalpha but not in PI3Kgamma (34). In contrast to the specificity of PI3Kgamma on shear stress-dependent activation of JNK in BAEC as shown in the present study, PI3Kgamma has been shown to act as an upstream regulator of both ERK and JNK in COS-7 cells (23, 24). These differences may be caused by different stimuli/receptor systems or cell types used.

The current findings raise several interesting questions regarding the mechanisms that link the early activation of PI3Kgamma to JNK activation some 30-60 min later. This relatively delayed activation of JNK is typical of many other stimuli including cytokines, various environmental stresses, hormones, and growth factors (1, 5, 21, 22, 25). Furthermore, in BAEC, brief exposure (<1 min) of cells to ultraviolet light or a strong oxidant, peroxynitrite, stimulates JNK activity 20-60 min after the treatments (data not shown). Shear stress activation of JNK is a further example of this delayed response. Interestingly, antioxidants such as N-acetyl cysteine have been shown to prevent JNK activation induced by many known JNK inducers (1, 21, 22, 25), suggesting a possible role of reactive oxygen species in the delayed response of JNK activation. Identification of the components linking the early signaling events to JNK activation is being actively pursued in our laboratories.

Currently, the physiological roles of PI3Kgamma and JNK in endothelial cells have not been defined. PI3K has been implicated in various cellular responses including antiapoptosis, vesicle transport, and actin cytoskeleton rearrangement (2, 8). It is interesting to note that shear stress also induces similar responses in endothelial cells. These responses include prevention of apoptosis induced by tissue necrosis factor-alpha , stimulation of pinocytosis, and rearrangement of actin cytoskeleton and cell shape (4, 7). Whether PI3Kgamma and JNK play a role in those mechanosensitive actions in endothelial cells needs to be determined.

    ACKNOWLEDGEMENTS

We thank Drs. A. Kraft and C. Franklin for providing a GST-c-Jun plasmid and a rabbit JNK antibody and Dr. M. P. Wymann for providing a PI3Kgamma plasmid.

    FOOTNOTES

This work was supported by National Institutes of Health First Award HL-53601 and American Heart Association Grant-In-Aid AL-G-960035 (to H. Jo).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: H. Jo, Dept. of Pathology, Div. of Molecular and Cellular Pathology, The Univ. of Alabama at Birmingham, G019C Volker Hall, Birmingham, AL 35294.

Received 3 June 1998; accepted in final form 7 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Assefa, Z., M. Garmyn, R. Bouillon, W. Merlevede, J. R. Vandenheede, and P. Agostinis. Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes. J. Invest. Dermatol. 108: 886-891, 1997[Medline].

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Am J Physiol Heart Circ Physiol 275(5):H1898-H1904
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