Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca2+ and Ca2+-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F2α. Results Pretreatment of the cells with the Ca2+ chelators BAPTA-AM or EGTA, as well as the Ca2+ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF2α. Conclusion The present data indicate that Ca2+ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.


Background
The extracellular signal regulated kinases ERK1 (p44 mapk ) and ERK2 (p42 mapk ) are activated in response to stimulation of receptor tyrosine kinases (RTKs) as well as heptahelical G protein coupled receptors (GPCR) and transmit signals which regulate cell differentiation and growth [1][2][3]. The molecular steps involved in signaling from GPCRs to ERK are incompletely understood. Data obtained in various cell systems have provided evidence in support of several signaling pathways including protein kinase C (PKC) [4], Ca 2+ -mediated mechanisms [5][6][7][8][9][10][11][12], and transactivation of receptor tyrosine kinases [13,14]. In hepatocytes several hormones, including vasopressin, angiotensin II, norepinephrine, and PGF 2α , that bind to GPCRs activate ERK [15][16][17]. The mechanisms mediating the ERK activation by GPCR agonists are not clarified; there is evidence that protein kinase C is involved [15,18], but a role for Ca 2+ also appears likely, since all the agents above activate phospholipase C and elevate intracellular Ca 2+ in hepatocytes [19,20]. Furthermore, agents that elevate intracellular Ca 2+ through mechanisms bypassing receptors have been found to activate ERK [15,21]. However, agonist-stimulated phospholipase C activity is rapidly down-regulated upon culturing of hepatocytes [22,23], and we recently reported that norepinephrine and PGF 2α activate ERK under conditions where the level of inositol 1,4,5-trisphosphate (InsP 3 ) was only slightly, and transiently elevated [17]. In the present study we have, therefore, examined more closely the role of Ca 2+ in ERK activation induced by norepinephrine and PGF 2α and mechanisms downstream of elevated [Ca 2+ ] i .

Agents that elevate [Ca 2+ ] i activate ERK
In agreement with previous observations [15,21] treatment of hepatocytes with thapsigargin, which inhibits Ca 2+ reuptake to endoplasmatic reticulum [24], and A23187, which induces Ca 2+ influx, stimulated ERK1/2 activity 2-2.5 fold (Fig. 1A). The elevation of intracellular Ca 2+ resulting from stimulation with thapsigargin is shown in Fig. 1B. These observations are compatible with a role for Ca 2+ -elevating mechanisms in the events that trigger ERK1/2 activation in hepatocytes. 2+ We then examined the role of Ca 2+ in activation of ERK1/ 2 induced by stimulation of α 1 -adrenoceptors (with norepinephrine in the presence of timolol) and prostaglandin receptors (using PGF 2α ) [21,25,26]. The hepatocytes were pretreated with BAPTA-AM, which is activated intracellularly to bind Ca 2+ , EGTA, which binds extracellular Ca 2+ and eventually may deplete intracellular Ca 2+ [27,28], or gadolinium, a competitive inhibitor of Ca 2+ influx [29][30][31]. BAPTA-AM completely attenuated the norepinephrine-induced rise of [Ca 2+ ] i ( Fig. 2A), while the ERK1/2 activity in response to norepinephrine was partially decreased (Fig. 2B,2C). ERK1/2 activity induced by PGF 2α and the Ca 2+ ionophore A23187 was also inhibited by BAPTA-AM, while the TPA response was unaffected (Fig.  2B,2C,2D). When the cells were pretreated with EGTA, the initial peak of the Ca 2+ elevation was only slightly affected, while the prolonged phase of the Ca 2+ -response was abolished (Fig. 3A). The activation of ERK1/2 by norepinephrine or PGF 2α was partly decreased by EGTA (Fig.  3B,3C,3D). EGTA also markedly decreased the ERK1/2 response induced by A23187 and thapsigargin, while the TPA-induced ERK1/2 activation was unaffected (Fig.  3B,3C). Pretreatment with gadolinium decreased the adrenergic activation almost to the level obtained by EGTA (Fig. 4A). Gadolinium also decreased the A23187induced activation of ERK1/2 (Fig. 4B). Taken together, the results suggest a role for Ca 2+ in the activation of ERK by norepinephrine and PGF 2α and that this involves Ca 2+ influx as well as release from internal pools.

Effect of antagonists of calmodulin and the multifunctional Ca 2+ /calmodulin-dependent protein kinase in ERK activation in hepatocytes
A major mechanism for Ca 2+ -induced signaling is through formation of a complex with calmodulin [32,33]. Calmodulin has been found to stimulate as well as inhibit ERK1/2 activity [12,34,35]. We therefore examined the role of calmodulin in these pathways. Pretreatment of hepatocytes with the calmodulin inhibitors trifluoperazine, J-8, and W-7 markedly inhibited the ERK1/2 activation after stimulation with norepinephrine and PGF 2α (Fig. 5). The results were confirmed with immunoblots ( Fig. 6). Activation of ERK1/2 by A23187 was also markedly sensitive to pretreatment with W-7 (Fig. 5, 6).
Calmodulin may act on several regulatory enzymes [32,[36][37][38][39][40], including the Ca 2+ /calmodulin-dependent protein kinases, which have been implicated in the activation of ERK1/2 [7][8][9]. We explored a possible role for the multifunctional Ca 2+ /calmodulin-dependent protein kinase in ERK1/2 activation in hepatocytes stimulated by norepinephrine and PGF 2α . Pretreatment of the cells with KN-93, an inhibitor of the multifunctional Ca 2+ /calmod-  ulin-dependent protein kinase [41], did not decrease the activation of ERK1/2 either by norepinephrine, PGF 2α , or A23187 (Fig. 5, 6). In supplementary experiments we examined the effects of higher concentrations of KN-93 (up to 100 µM) or prolonged exposure times (up to 24 hours) which in none of the cases resulted in a decreased ERK1/2 activation (data not shown). These results suggest that calmodulin might be involved in hormone-induced activation of ERK1/2 in hepatocytes, however the data do not support a role for the multifunctional Ca 2+ /calmodulindependent protein kinase in Ca 2+ /calmodulin-mediated activation of ERK1/2 in hepatocytes

Inhibitors of src kinases attenuate ERK activation in hepatocytes
Src kinases [42] have been implicated in the mechanisms resulting in ERK1/2 activation in response to stimulation of both G i -and G q -coupled heptahelical receptors [43][44][45], and several observations suggest that activation of Src in these pathways involves Ca 2+ [46,47]. Data obtained in this study showed that the Src inhibitors PP1 and PP2, which are reported to primarily inhibit the Lck, Fyn, and Hck subtypes of Src kinases [48], markedly decreased the PGF 2α -induced ERK1/2 activation and led to partial inhibition of the effect of norepinephrine, while the EGF induced ERK1/2 response was not reduced (Fig. 7). Furthermore, ERK1/2 activation induced by A23187 and . The results suggest a role for Src kinases in the mechanisms leading to ERK1/2 activation both by PGF 2α and norepinephrine, and that this step at least in part may be located distal to increases in the intracellular level of Ca 2+ .

Discussion
The present findings confirm previous reports of a role for Ca 2+ in ERK1/2 activation in hepatocytes [15,21] and suggest that release of Ca 2+ from intracellular stores as well as influx of extracellular Ca 2+ is of importance for the hormone-induced activation of ERK1/2. Furthermore, the results suggest that calmodulin and Src kinases might be involved in the Ca 2+ -dependent activation of ERK1/ERK2.
The present data suggest that Ca 2+ is involved in activation of ERK1/2 in hepatocytes in response to norepinephrine and PGF 2α . The ERK1/2 response was decreased by chelation of intracellular and extracellular Ca 2+ with BAP-TA-AM and EGTA, respectively, as well as by gadolinium, which competitively inhibits Ca 2+ influx. It may appear that extracellular and intracellular Ca 2+ act in a concerted, possibly sequential manner in the mechanisms involved in activation of ERK1/2 by norepinephrine and PGF 2α . An integration of Ca 2+ signals from the extracellular and intracellular environment is presumably due to store-operated Ca 2+ influx [31,55,56]. The mechanisms that initiate Ca 2+ influx subsequent to depletion of intracellular stores are incompletely understood, but recent studies have suggested that direct interaction between InsP 3 receptors and calcium channels in the plasma membrane may lead to activation of the calcium channels [57]. A diffusible Ca 2+ influx factor may also be involved [58]. Previous studies have suggested that hormone-induced Ca 2+ influx involves heterotrimeric G i proteins in hepatocytes [59,60]. It is notable that norepinephrine and PGF 2α activate ERK1/2 in the presence of a barely detectable increase in intracellular InsP 3 [17]. This may suggest either the occurrence of local elevations of InsP 3 which do not affect global InsP 3 , or that Ca 2+ pools are regulated by other mechanisms such as generation of sphingosine-1-phos- phate [61]. A role for ryanodine-sensitive Ca 2+ stores in the endoplasmatic reticulum has also been proposed in hepatocytes [62] and EGTA-sensitive pools that are located in plasma membrane micro villar structures have been described [63].
Our data further suggest that calmodulin, which has previously been implicated in growth regulation in liver [64], is involved in activation of ERK1/2. The ERK1/2 responses induced by norepinephrine and PGF 2α were markedly decreased after pretreatment with the calmodulin inhibitors trifluoperazine, J-8, or W-7. Besides calmodulin, it is conceivable that the effect of Ca 2+ is mediated through other Ca 2+ -binding proteins [39,65]. Calmodulin may also act in a Ca 2+ -independent manner [38,66], which might account for the more pronounced inhibition of hormonestimulated ERK activity by calmodulin antagonists than by agents inhibiting the Ca 2+ signal. Alternatively, nonspecific effects produced by calmodulin antagonists in higher doses might explain their relatively stronger inhibition. Among the downstream targets of calmodulin, the Ca 2+ /calmodulin-dependent protein kinases have been implicated in ERK1/2 activation in smooth muscle cells [8,9], but not in other cells [67,68]. Furthermore, the multifunctional Ca 2+ /calmodulin-dependent protein kinase was located downstream of ERK1/2 activation by plateletderived growth factor (PDGF) in vascular smooth muscle cells [69]. Pretreatment of hepatocytes with KN-93 did not decrease ERK1/2 activation induced by hormones or the Ca 2+ ionophore A23187. Thus, while the multifunctional Ca 2+ /calmodulin-dependent protein kinase exerts several effects in hepatocytes, including growth inhibition under certain conditions [70][71][72], it does not appear to be involved in ERK activation. It is of interest that the α 1 -adrenoceptor-induced c-fos expression in fibroblasts was also observed to involve calmodulin, but not the multifunctional Ca 2+ /calmodulin-dependent protein kinase [73].
Increasing evidence suggest a role of Src kinases downstream of Ca 2+ /calmodulin in ERK1/2 signaling [10,14,46]. The present results suggest that Src kinases may be involved in ERK1/2 activation induced by PGF 2α and norepinephrine, while the EGF induced ERK1/2 response appears to be independent of these Src kinases. Furthermore, the ERK1/2 activation induced by the Ca 2+ ionophore A23187 or by thapsigargin was partially decreased by Src inhibition suggesting a role of Src distal to increases in intracellular Ca 2+ . Of the possible downstream targets for Ca 2+ /calmodulin in ERK signaling in hepatocytes our findings thus lend support to a role of Src kinases, although the results do not exclude the possibility that Src kinases and calmodulin act in parallel pathways leading to ERK activation.
While the present results show a role for Ca 2+ in ERK1/2 activation by norepinephrine and PGF 2α , it is notable that even complete inhibition of Ca 2+ signaling only partially inhibited ERK1/2 activity. Taken together with previous observations that inhibition of PKC almost completely inhibited ERK1/2 activation by norepinephrine, vasopressin, and angiotensin II [18], the results suggest that several mechanisms contribute to and may act in concert in the hormonal stimulation of ERK1/2 in hepatocytes.

Conclusion
Our present data indicate that both extracellular and intracellular Ca 2+ is involved in hormone-induced ERK1/2 activation in cultured hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways, while the multifunctional Ca 2+ / calmodulin-dependent protein kinase does not appear to be involved.

Isolation and culture of hepatocytes
Male Wistar rats (170-220 g) fed ad libitum were used. Parenchymal liver cells were isolated by in vitro collagenase perfusion and low-speed centrifugation [74] with modifications as previously described [75]. Cell viability was at least 95 %, measured as the ability to exclude trypan blue. The cells were suspended in medium and plated in Costar wells at 20.000 cells/cm 2 , unless otherwise specified. The culture medium (0.2 ml/cm 2 ) was a 1:1 mixture of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 containing 16.8 mM glucose [76], supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), dexamethasone (25 nM) and insulin (100 nM). The cultures were gassed with 95 % air/ 5 % CO 2 and kept at 37°C.

Measurement of ERK activity
The measurement of ERK1/2 activity was performed as previously described [17,77]. In brief, the hepatocyte cultures were exposed to agonists for 5 minutes before rinsing and scraping the cells into a 10 % ethylene glycol buffer. The lysate was centrifugated (15,800 × g) for 10 min, and the supernatant was mixed with phenyl-Sepharose which was washed twice in a 10 %, twice in a 35 % ethylene glycol buffer, and finally ERK1/2 was eluted with a 60 % ethylene glycol buffer [78]. The eluate was assayed for ERK1/2 activity with MBP as substrate, thereafter spotted onto P81 paper (Whatman, Maidstone, UK), which was washed, dried and counted in a liquid scintillation counter. Protein content was determined with the BCA Protein Assay (Pierce, Rockford, IL, U.S.A.).

Immunoblotting
Aliquots with 20 µg cell protein (total cell lysate prepared in Laemmli buffer) were electrophoresed on 10 % polyacrylamide gels (acrylamide:N'N'-bis-methylene acrylamide 30:0.8) followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with a polyclonal ERK1/2 antibody against the dually threonineand tyrosine phosphorylated forms of ERK1 and ERK2 (Promega Corporation, Madison, WI). Assessment of the multifunctional Ca 2+ /calmodulin dependent protein kinase was performed by immunoblotting using an antibody against the phosphorylated from of the enzyme. Immunoreactive bands were visualised with ECL Western blotting detection reagents (Amersham International).