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Phosphatidylinositol 3-Kinase Mediates the Inhibitory Effect of Epidermal Growth Factor on Calcium-dependent Chloride Secretion*

  • Jorge M. Uribe
    Footnotes
    Affiliations
    Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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  • Stephen J. Keely
    Affiliations
    Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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  • Alexis E. Traynor-Kaplan
    Footnotes
    Affiliations
    Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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  • Kim E. Barrett
    Correspondence
    Faculty member in the Biomedical Sciences Dept. of UCSD School of Medicine. To whom correspondence should be addressed:
    Affiliations
    Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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  • Author Footnotes
    * These studies were supported by Grant DK28305 (to K. E. B.) from the National Institutes of Health. 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. Section 1734 solely to indicate this fact.
    Recipient of a Predoctoral Fellowship from Institutional Training Grant DK07202 in Digestive Diseases. Predoctoral student in the Biomedical Sciences Ph.D. program of UCSD School of Medicine.
    § Recipient of an AGA/Fiterman award and Grant DK47240 from the National Institutes of Health. Faculty member in the Biomedical Sciences Dept. of UCSD School of Medicine.
Open AccessPublished:October 25, 1996DOI:https://doi.org/10.1074/jbc.271.43.26588
      Epidermal growth factor (EGF) and carbachol both inhibit calcium-activated chloride secretion by the human colonic epithelial cell line, T84. Although the inhibitory mechanism for the carbachol effect involves the 3,4,5,6-isomer of inositol tetrakisphosphate, the mechanisms responsible for the EGF effect have not yet been fully elucidated. Here, we studied the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the inhibitory effect of EGF. The PI 3-kinase inhibitor, wortmannin, slightly increased basal chloride secretion and potentiated the secretory response to thapsigargin. Wortmannin also partially reversed EGF-induced, but not carbachol-induced, inhibition of thapsigargin-stimulated chloride secretion. Wortmannin alone had no effect on carbachol- or histamine-induced chloride secretion and completely reversed EGF-induced inhibition of the secretory response to these agonists. EGF, carbachol, histamine, and thapsigargin all increased levels of the 85-kDa regulatory subunit of PI 3-kinase in antiphosphotyrosine immunoprecipitates. However, only EGF significantly increased levels of the 110-kDa catalytic subunit. Furthermore, only EGF increased PI 3-kinase activity in an in vitro kinase assay. High levels of phosphatidylinositol (
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )-monophosphate were present in unstimulated cells and significantly reduced by wortmannin. EGF, but not carbachol, rapidly increased levels of phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate. Production of these lipids was also sensitive to wortmannin. Our data suggest that EGF activates PI 3-kinase and that its lipid products may mediate the inhibitory effect of EGF on calcium-dependent chloride secretion. Our data also suggest that a phosphatidylinositol-specific 3-kinase activity is present in unstimulated T84 cells and may regulate production of phosphatidylinositol (
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )-monophosphate and basal secretory tone.

      INTRODUCTION

      The regulation of intestinal epithelial chloride transport is under the control of neural, humoral, and immune-related mechanisms (
      • Barrett K.E.
      ). As the active transepithelial movement of chloride into the gastrointestinal lumen is an important mechanism governing the passive movement of water, breakdown in the regulation of this process can lead to excessive secretory responses, resulting in diarrhea. Our laboratory has focused on understanding the intracellular mechanisms responsible for the regulation of chloride secretion.
      Chloride secretion is positively regulated via two predominant pathways, utilizing cyclic nucleotides or calcium as second messengers, respectively (
      • Barrett K.E.
      ). Calcium-dependent secretion can be evoked experimentally by agents such as the muscarinic agonist, carbachol, histamine, or the calcium ATPase inhibitor, thapsigargin. All of these stimuli elevate cytoplasmic calcium concentrations, which in turn evokes secretion. Moreover, we have shown that calcium-dependent chloride secretion is also subject to a number of negative regulatory influences. Thus carbachol, while itself serving as an initial agonist of chloride secretion, can also subsequently inhibit calcium-dependent chloride secretion, such as that stimulated by thapsigargin (
      • Kachintorn U.
      • Vajanaphanich M.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      ). This inhibition occurs at a step distal to the rise in intracellular calcium and has been attributed to a putative negative messenger, inositol(3,4,5,6)tetrakisphosphate (D-Ins(3,4,5,6)P4)
      The abbreviations used are: Ins(3,4,5,6)P4
      inositol (3,4,5,6)-tetrakisphosphate
      EGF
      epidermal growth factor
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PtdIns 3-kinase
      phosphatidylinositol-specific 3-kinase
      p85
      85-kDa subunit of phosphatidylinositol 3-kinase
      p110
      110-kDa subunit of phosphatidylinositol 3-kinase
      PI(
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )P, phosphatidylinositol (
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )-monophosphate, PI(3,4)P2
      phosphatidylinositol (3,4)-bisphosphate
      PI(3,4,5)P3
      phosphatidylinositol (3,4,5)-trisphosphate
      SH
      src homology
      Isc
      short circuit current
      PBS
      phosphate-buffered saline
      PKC
      protein kinase C.
      (
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      ). This inositol polyphosphate may directly block calcium-activated chloride channels (
      • Ismailov I.I.
      • Fuller C.M.
      • Berdiev B.K.
      • Shlyonsky V.G.
      • Benos D.J.
      • Barrett K.E.
      ), thereby inhibiting the overall process of transepithelial chloride secretion. The peptide growth factor, epidermal growth factor (EGF), also exerts an inhibitory effect on calcium-dependent chloride secretion but does not itself act as a secretagogue (
      • Uribe J.M.
      • Gelbmann C.M.
      • Traynor-Kaplan A.E.
      • Barrett K.E.
      ). The inhibitory effect of EGF displays some similarities to that evoked by carbachol. Thus, EGF inhibits calcium-activated secretion without affecting the rise in intracellular calcium. EGF also causes a comparatively small increase in dl-Ins(3,4,5,6)P4 (
      • Uribe J.M.
      • Gelbmann C.M.
      • Traynor-Kaplan A.E.
      • Barrett K.E.
      ). However, we recently demonstrated that this likely does not account for the inhibitory effects of EGF on secretion.
      J. M. Uribe, A. E. Traynor-Kaplan, and K. E. Barrett, submitted for publication.
      Moreover, simultaneous addition of maximally inhibitory concentrations of EGF and carbachol had a greater inhibitory effect on chloride secretion compared with either agent alone (
      • Uribe J.M.
      • Gelbmann C.M.
      • Traynor-Kaplan A.E.
      • Barrett K.E.
      ). These data suggest that EGF is activating a novel mechanism responsible for the inhibition of calcium-dependent chloride secretion that is distinct from that evoked by carbachol.
      Our laboratory has demonstrated a relationship between tyrosine phosphorylation and inhibition of chloride secretion (
      • Keely S.J.
      • Uribe J.M.
      • Barrett K.E.
      ). Therefore, we hypothesized that the activation of tyrosine kinase-dependent pathways by EGF is involved in the inhibition of chloride secretion. One signaling pathway regulated by tyrosine phosphorylation in response to EGF, in other cell types, involves the recruitment of phosphatidylinositol 3-kinase (PI 3-kinase) (
      • Kapellar R.
      • Cantley L.C.
      ). This lipid kinase is responsible for the production of D-3-phosphorylated phosphoinositides. Its activation involves recruitment of the SH2 domains of its 85-kDa regulatory subunit to tyrosine-phosphorylated residues of receptor proteins (erbB3, p120cbl) (
      • Soltoff S.P.
      • Carraway K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ), thus bringing the associated 110-kDa catalytic subunit closer to its membrane substrates. Furthermore, in vitro binding of p85 to tyrosine-phosphorylated residues enhances the activity of p110 (
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ). The activation of PI 3-kinase and consequent generation of its lipid products has been implicated in cell growth (
      • Valius M.
      • Kazlauskas A.
      ), movement (
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.
      • Heldin C.
      • Mori S.
      • Claesson-Welsh L.
      ), vesicular transport (
      • Martys J.L.
      • Wjasow C.
      • Gangi D.M.
      • Kielian M.C.
      • McGraw T.E.
      • Backer J.M.
      ), glucose uptake (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ), and oxidant production (
      • Ahmed M.U.
      • Hazeki K.
      • Hazeki O.
      • Katada T.
      • Ui M.
      ). Recently, PI 3-kinase has been reported to regulate the activity of the Na+/H+ exchanger (
      • Ma Y.
      • Reusch H.P.
      • Wilson E.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      • Ives H.E.
      ,
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.
      • Donowitz M.
      ). This led us to speculate that PI 3-kinase might also regulate other epithelial transport proteins, such as those involved in chloride secretion. Thus, we sought to determine if EGF activates PI 3-kinase in T84 cells, and, if so, its involvement in inhibition of calcium-dependent chloride secretion by EGF. Furthermore, carbachol has also been shown to increase the amount of tyrosine-phosphorylated proteins in T84 cells (
      • Keely S.J.
      • Uribe J.M.
      • Barrett K.E.
      ). We therefore additionally wanted to determine if carbachol also activates PI 3-kinase and mediates any of its inhibitory effects through activation of this enzyme. Finally, a constitutively active phosphatidylinositol-specific 3-kinase (PtdIns 3-kinase) has been detected in yeast (
      • Herman P.K.
      • Emr S.
      ), and a homologue of this protein may also be present in mammalian cells (
      • Volinia S.
      • Dhand R.
      • Vanhaesebroeck B.
      • MacDougall L.K.
      • Stein R.
      • Zvelebil M.J.
      • Domin J.
      • Panaretou C.
      • Waterfield M.D.
      ). Thus, we finally wanted to determine if PI 3-kinase activity plays any role in regulating basal chloride secretion.

      DISCUSSION

      PI 3-kinase is a lipid kinase responsible for the production of 3-phosphorylated lipids and has been implicated in cell proliferation (
      • Valius M.
      • Kazlauskas A.
      ), cell movement (
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.
      • Heldin C.
      • Mori S.
      • Claesson-Welsh L.
      ), and glucose transport (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). The activation of PI 3-kinase has also been shown to be involved in regulating the Na+/H+ exchanger (
      • Ma Y.
      • Reusch H.P.
      • Wilson E.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      • Ives H.E.
      ) and perhaps in the activation of NaCl absorption (
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.
      • Donowitz M.
      ). It is often the case that pathways involved in the activation of intestinal secretion simultaneously decrease absorption. By analogy, we hypothesized that PI 3-kinase might be involved in mediating the inhibitory effect of EGF on chloride secretion. In fact, the ability of wortmannin, a PI 3-kinase inhibitor, to reverse inhibitory actions of EGF indeed suggests involvement of PI 3-kinase in this process. Thus, wortmannin partially reversed EGF-induced inhibition of thapsigargin-stimulated chloride secretion and completely reversed EGF's inhibition of carbachol- and histamine-induced chloride secretion.
      The fact that wortmannin only partially reversed the inhibitory effect of EGF on thapsigargin-induced secretion merits some comment. On the surface, these results are difficult to reconcile with the fact that carbachol, histamine, and thapsigargin all activate calcium-dependent chloride secretory responses. However, the secretory response to thapsigargin was potentiated by wortmannin alone while those to carbachol and histamine were not. Chloride secretion evoked by thapsigargin appears to involve only the elevation of intracellular calcium (
      • Kachintorn U.
      • Vajanaphanich M.
      • Traynor-Kaplan A.E.
      • Dharmsathaphorn K.
      • Barrett K.E.
      ), whereas G-protein-mediated agonists such as carbachol and histamine likely activate additional signaling pathways (
      • Dharmsathaphorn K.
      • Cohn J.
      • Beuerlein G.
      ). Both carbachol and histamine evoke responses that are more transient than that induced by thapsigargin. Thus, we hypothesize that they may be activating other inhibitory pathways that may override any potentiative effects wortmannin could exert.
      EGF induced a rapid and sustained elevation of PI(3,4)P2 and PI(3,4,5)P3 in T84 cells. This effect correlates well with the ability of EGF to inhibit calcium-activated chloride secretion. EGF inhibits carbachol-induced chloride secretory responses within 1 min, and the inhibition is then maintained for at least 60 min (
      • Uribe J.M.
      • Gelbmann C.M.
      • Traynor-Kaplan A.E.
      • Barrett K.E.
      ). In addition, the increase in these lipids induced by EGF was completely inhibited by wortmannin, further suggesting a role for these lipids in EGF inhibition of secretion. However, the mechanism whereby these lipids might inhibit secretion is currently not known. Preliminary data suggest that EGF exerts a wortmannin-sensitive inhibitory effect upon a basolateral potassium channel (
      • Smitham J.
      • Uribe J.M.
      • Barrett K.E.
      ). Furthermore, previous studies have shown that the basolateral potassium channel involved in calcium-activated chloride secretion is inhibited by protein kinase C (PKC) (
      • Reenstra W.W.
      ). 3-Phosphorylated phospholipids have recently been demonstrated to be capable of activating both novel and atypical PKCs in both in vitro and in vivo systems (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.-I.
      • Mizuno K.
      • Hirai S.-I.
      • Kazlauskas A.
      • Ohno S.
      ). Preliminary data from our lab also suggest that these novel and atypical isoforms of PKC are present in T84 cells.
      J. M. Uribe and K. E. Barrett, unpublished observations.
      Thus, 3-phosphorylated phospholipids could activate these PKCs in the process of inhibiting chloride secretion. As these isoforms of PKC are quite insensitive to available inhibitors (
      • Seynaeve C.M.
      • Kazanietz M.G.
      • Blumberg P.M.
      • Sausville E.A.
      • Worland P.J.
      ), further experiments, utilizing more sophisticated molecular approaches, will be required to test this hypothesis.
      We also observed a small elevation in basal chloride secretion in response to wortmannin, which correlated with a reduction in the basal levels of PI(
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )P. Recently, a mammalian PtdIns 3-kinase activity was found that is highly homologous to yeast Vps34p (
      • Volinia S.
      • Dhand R.
      • Vanhaesebroeck B.
      • MacDougall L.K.
      • Stein R.
      • Zvelebil M.J.
      • Domin J.
      • Panaretou C.
      • Waterfield M.D.
      ). The existence of this isoform of PI 3-kinase, and the presence of PI 3-kinase activity in antiphosphotyrosine immunoprecipitates from unstimulated cells, may explain the relatively high levels of PI(
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )P in resting T84 cells. In other systems, wortmannin has been shown to affect basal endocytotic processes and vesicular trafficking, with these effects attributed to actions on PI 3-kinase activity and 3-phosphorylated lipids (
      • Martys J.L.
      • Wjasow C.
      • Gangi D.M.
      • Kielian M.C.
      • McGraw T.E.
      • Backer J.M.
      ). We can therefore speculate that wortmannin, by reducing basal levels of PI(
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )P, might also regulate the trafficking of various membrane proteins involved in chloride secretion. Trafficking of membrane proteins clearly plays a role in regulating secretion, since cAMP-mediated secretory responses, for example, require the insertion of Na+/K+/2Cl cotransporters into the basolateral membrane for maximal activity (
      • Matthews J.B.
      • Awtrey C.S.
      • Madara J.L.
      ). Thus, altering the levels of certain membrane proteins could also conceivably result in negative regulation of secretion.
      Some studies suggest that wortmannin may not be wholly specific for the inhibition of PI 3-kinase. Inhibition of myosin light chain kinase (
      • Nakanishi S.
      • Kakita S.
      • Takahashi I.
      • Kawahara K.
      • Tsukuda E.
      • Sano T.
      • Yamada K.
      • Yoshida M.
      • Kase H.
      • Matsuda Y.
      • Hashimoto Y.
      • Nonomura Y.
      ), PI 4-kinase (
      • Nakanishi S.
      • Catt K.J.
      • Balla T.
      ), phospholipase A2 (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.O.
      ), and phospholipase D (
      • Reinhold S.L.
      • Prescott S.M.
      • Zimmerman G.A.
      • McIntyre T.M.
      ) have also been reported. However, at the concentrations of wortmannin used in our study (50 nM), inhibition of myosin light chain kinase is not likely, and no reduction in the level of PI(
      • Ismailov I.I.
      • Fuller C.M.
      • Berdiev B.K.
      • Shlyonsky V.G.
      • Benos D.J.
      • Barrett K.E.
      )P was observed. It is also unlikely that wortmannin is inhibiting phospholipase A2 or phospholipase D in our system since their products, arachidonic acid and phosphatidic acid, respectively, have pro-secretory effects (
      • Barrett K.E.
      • Bigby T.D.
      ,
      • Vajanaphanich M.
      • Kachintorn U.
      • Barrett K.E.
      • Cohn J.A.
      • Dharmsathaphorn K.
      • Traynor-Kaplan A.E.
      ). Inhibition of these enzymes would therefore be expected to reverse a stimulatory rather than an inhibitory effect on secretion. We therefore believe that the actions of wortmannin on chloride secretion are likely reflective of its specific inhibition of PI 3-kinase.
      The significance of the ability of carbachol, histamine, and thapsigargin to recruit p85, independent of p110, is currently unknown. p85 contains an SH3 domain, a proline-rich sequence, and a region homologous to the bcr gene product, in addition to its 2 SH2 domains. It is therefore likely that p85 can couple proteins to tyrosine kinase-dependent signaling pathways, in a manner similar to the adaptor proteins, Grb2 and Shc (
      • Keely S.J.
      • Uribe J.M.
      • Barrett K.E.
      ). Studies have in fact shown that p85 can be recruited to tyrosine-phosphorylated proteins independent of p110 (
      • Sung C.K.
      • Sanchez-Margalet V.
      • Goldfine I.D.
      ). Other studies have demonstrated that p85 is able to couple to p120 Gap and Grb2 (
      • DePaolo D.
      • Reusch J.E.-B.
      • Carel K.
      • Bhuripanyo P.
      ,
      • Wang J.
      • Auger K.R.
      • Jarvis L.
      • Shi Y.
      • Roberts T.M.
      ), proteins involved in the regulation of Ras activation. As carbachol has been shown to activate Ras in other cell systems (
      • Winitz S.
      • Russell M.
      • Qian N.-X.
      • Gardner A.
      • Dwyer L.
      • Johnson G.L.
      ), it is possible that calcium agonists activate the Ras pathway via coupling to p85. However, a definitive answer to this question, as well as an understanding of its significance for cell function, will require further study.
      In summary, we have shown that EGF can activate PI 3-kinase and that the lipid products of this enzyme may regulate calcium-activated chloride secretion. Furthermore, our data suggest that a PtdIns 3-kinase activity is present in untreated T84 cells and that this activity, through the production of PI(
      • Vajanaphanich M.
      • Schultz C.
      • Rudolf M.
      • Wasserman M.
      • Enyedi P.
      • Craxton A.
      • Shears S.
      • Tsien R.
      • Barrett K.E.
      • Traynor-Kaplan A.E.
      )P, may be involved in the regulation of basal chloride secretory tone. We speculate that, in addition to its mitogenic effects, the activation of PI 3-kinase may be involved in growth factor-induced effects on the epithelium that could reflect adaptive responses to epithelial damage and inflammation. By limiting active secretion, cellular energy resources could then be diverted to cell proliferation and epithelial restitution, while limiting inappropriate fluid and electrolyte loss. Finally, we have shown that the inhibitory effects of carbachol on chloride secretion likely do not involve PI 3-kinase activity. However, the ability of carbachol and histamine to recruit either p85 alone, or small amounts of the p85/p110 complex, without activating PI 3-kinase, may have alternate signaling consequences that have yet to be elucidated.

      Acknowledgments

      We thank Ginger Westbrook for assistance with manuscript preparation.

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