Requirement for Protein Kinase C θ for Cell Cycle Progression and Formation of Actin Stress Fibers and Filopodia in Vascular Endothelial Cells

Abstract

Activation of the protein kinase C (PKC) family with phorbol esters induces endothelial proliferation and angiogenesis, but which of the events that constitute angiogenesis are affected by individual members of the PKC family is unknown. In rat capillary endothelial (RCE) cells, serum stimulation increased expression of a single PKC isoenzyme, PKCθ, and its translocation to the periphery. Conditional overexpression of a dominant-negative mutant of PKCθ markedly inhibited RCE proliferation, as well as closure of a “wound” by RCE migration and formation of capillary rings and tubules in vitro. PKCθ inhibition delayed the endothelial cell cycle at the G2/M phase and prevented formation of actin stress fibers and filopodia but not lamellipodia. The defect in cell morphology and wound closure in PKCθ-kn cells was reversed by overexpressing kinase-active PKCθ, indicating that these RCE functions depend upon PKCθ substrates. Thus, PKCθ is required for multiple processes essential for angiogenesis and wound repair, including endothelial mitosis, maintenance of a normal actin cytoskeleton, and formation of an enclosed tube. Activation of the protein kinase C (PKC) family with phorbol esters induces endothelial proliferation and angiogenesis, but which of the events that constitute angiogenesis are affected by individual members of the PKC family is unknown. In rat capillary endothelial (RCE) cells, serum stimulation increased expression of a single PKC isoenzyme, PKCθ, and its translocation to the periphery. Conditional overexpression of a dominant-negative mutant of PKCθ markedly inhibited RCE proliferation, as well as closure of a “wound” by RCE migration and formation of capillary rings and tubules in vitro. PKCθ inhibition delayed the endothelial cell cycle at the G2/M phase and prevented formation of actin stress fibers and filopodia but not lamellipodia. The defect in cell morphology and wound closure in PKCθ-kn cells was reversed by overexpressing kinase-active PKCθ, indicating that these RCE functions depend upon PKCθ substrates. Thus, PKCθ is required for multiple processes essential for angiogenesis and wound repair, including endothelial mitosis, maintenance of a normal actin cytoskeleton, and formation of an enclosed tube. Proliferation and migration of vascular endothelial cells are essential features of angiogenesis and wound repair (1Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 2Jackson D. Volpert O.V. Bouck N. Linzer D.I. Science. 1994; 266: 1581-1584Crossref PubMed Scopus (206) Google Scholar). Both of these processes, as well as formation of a vessel tube, require tightly regulated changes in the endothelial actin cytoskeleton. Upon cell contact, proliferating endothelial cells become quiescent and develop a static actin cytoskeleton, a prominent feature of which is a peripheral actin band (3Coomber B.L. Gotlieb A.I. Arteriosclerosis. 1990; 10: 215-222Crossref PubMed Google Scholar, 4Wong M.K.K. Gotlieb A. Arteriosclerosis. 1990; 10: 76-84Crossref PubMed Google Scholar). When contact inhibition is released, such as occurs during vascular injury or following the matrix dissolution that accompanies angiogenesis, endothelial cells lose this peripheral actin band, reorganize the actin filaments, reenter the cell cycle, and proliferate. Structures that often develop in migrating fibroblasts include actin stress fibers, filopodia, and lamellipodia, or ruffling (5Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3771) Google Scholar, 6Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3104) Google Scholar). Some, if not all, of these actin structures also occur in endothelial cells stimulated in vitro (7Hoock T.C. Newcomb P.M. Herman I.M. J. Cell Biol. 1991; 112: 654-664Crossref Scopus (187) Google Scholar) or in vivo (8White G.E. Gimbrone Jr., M.A. Fujiwara K. J. Cell Biol. 1983; 97: 416-424Crossref PubMed Scopus (164) Google Scholar, 9Wong A.J. Pollard T.D. Herman I.M. Science. 1983; 219: 867-869Crossref PubMed Scopus (255) Google Scholar). Although several growth factors, cytokines, and their receptors that are required or sufficient for endothelial migration, proliferation, and angiogenesis have been identified (10Coomber B.L. Exp. Cell Res. 1991; 194: 42-47Crossref PubMed Scopus (40) Google Scholar, 11Fan T.-P.D. Jaggar R. Bicknell R. Trends Pharmacol. Sci. 1995; 16: 57-66Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 12Folkman J. N. Engl. J. Med. 1995; 333: 1757-1763Crossref PubMed Scopus (2260) Google Scholar), the intracellular messengers that mediate these processes are largely unrecognized. One group of mediators that are thought to participate in wound repair and angiogenesis are those in the protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C, RCE, rat capillary endothelial; PBS, phosphate-buffered saline; kn, kinase-negative; BSA, bovine serum albumin; FBS, fetal bovine serum. family. In some experimental models, addition of phorbol esters, which activate several members of this family, promote endothelial proliferation, migration, and angiogenesis, and inhibition of PKC with chemical inhibitors prevents growth and angiogenesis to at least some mitogens (13Daviet I. Herbert J.M. Maffrand M. Biochem. Biophys. Res. Commun. 1989; 158: 584-589Crossref PubMed Scopus (18) Google Scholar, 14Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1227) Google Scholar, 15Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (69) Google Scholar, 16Presta M. Tiberio L. Rusnati M. Dell'Era P. Ragnotti G. Cell Regul. 1991; 199: 719-726Crossref Scopus (58) Google Scholar). Conversely, others have observed inhibitory effects on endothelial growth following activation of PKC, depending upon the experimental conditions (17Doctrow S.R. Folkman J. J. Cell Biol. 1987; 104: 679-687Crossref PubMed Scopus (67) Google Scholar, 18Kosaka C. Sasaguri T. Ishida A. Ogata J. Am. J. Physiol. 1996; 270: C170-C178Crossref PubMed Google Scholar). Part of the difficulty in determining the mechanism of either of these phenomena is that the available tools (chemical inhibitors, phorbol esters) inhibit or activate many of the individual gene products that comprise the PKC family; results using these methods reflect the activity of more than one, possibly competing, pathway. Each of these members are separate gene products that have distinct cofactors and substrates (19Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3543) Google Scholar) and subserve distinct intracellular functions (20Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar, 21Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar); thus, it seems possible that they mediate different events in endothelial growth. The purpose of this study was to determine whether an individual isoenzyme of PKC is required for endothelial cell proliferation and angiogenesis and to discern the mechanism by which it acts. In rat capillary endothelial cells (RCE), initial experiments revealed that stimulation with serum increased expression of PKCθ but did not change expression of the other phorbol ester-sensitive isoenzymes. Thus, the possibility that PKCθ might mediate one or more steps in endothelial migration and proliferation was suggested and further investigated in this study. The full-length cDNA of the human PKCθ (22Baier G. Telford D. Giampa L. Coggeshall K.M. Baier-Bitterlich G. Isakov N. Altman A. J. Biol. Chem. 1993; 268: 4997-5004Abstract Full Text PDF PubMed Google Scholar, 23Chang J.D. Xu Y. Raychowdhury M.K. Ware J.A. J. Biol. Chem. 1993; 268: 14208-14214Abstract Full Text PDF PubMed Google Scholar) with a sequence encoding an amino-terminal fusion Flag epitope was cloned into the plasmid pGRE5-1 containing a glucocorticoid-inducible promoter (a gift of Dr. J.H. White, Montreal University; Ref. 24Mader S. White J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5603-5607Crossref PubMed Scopus (82) Google Scholar), using the EcoRI (5′-end) and BglII (3′-end) restriction endonucleotide enzyme sites, along with a neomycin resistance gene. Mutations in the regulatory domain and the kinase domain were synthesized within EcoRI-AflII-(1003) or AflII-(1003)-BglII cassettes, respectively, using the polymerase chain reaction method. To construct a constitutively active form of PKCθ, Arg-145 and Arg-146 in the regulatory domain were replaced with Ile and Trp. This mutation prevents the pseudosubstrate sequence from inhibiting the kinase domain and results in a PKC that does not require lipid activators to phosphorylate its substrate or to translocate to the surface membrane (25Pears G. C. J. Biochem. 1990; 194: PubMed Scopus Google Scholar). in the of the kinase domain was replaced with to a PKCθ protein that with the form for as for other PKC I. L. J. J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, S. Y. Y. A. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, J.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). in the kinase domain of other have upon the to but not its substrates and to as a dominant-negative J.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the sequence of construct was to the of the mutation and to for The were into rat capillary endothelial (RCE) cells (a gift of D. Ref. A.I. J. Cell Biol. 1990; PubMed Scopus Google Scholar) using the The RCE cells were in with fetal bovine serum and that the or its PKCθ were by resistance to To construct a plasmid for in the or experiments the gene in the plasmid was and and was into and of PKCθ to become constitutively active as cDNA was cloned into the plasmid as the cells were in the which of of that did not cell morphology or of normal RCE was to the to expression of the cloned The of the PKCθ or its was by and are with and are of results with other in which the were Cell were by addition of of were on a and to The were with either or Flag in containing and of RCE cells were with and with phosphate-buffered The cell was in and on for The were the was with an of and was to the and at for was with with and in of was to on a and to membrane to the The was and with cDNA at for in were conditions and to of RCE cells were with and and with phosphate-buffered cells from were in of for and were by a Cell was by at at for of or bovine serum as was to the and for at protein was and at with The were by at for at and with The was in of and in the kinase The kinase activity was using a C of and phorbol and PKC substrate of and of was to and for at In the to the activity of the constitutively active was to the The reaction was with of were used to separate The were with and were to for The of PKCθ in the was by with either or Cell growth was by the cells with a cells were in for and at in of for the RCE were with PBS, with and in the for RCE cells were on in in In some serum was from the to The was by with a The of the was a phase of a with a and with a and were in following the RCE cells were with and to cells were on the of with The were a capillary or a phase of a with a of PKCθ, cells on were with and were with in for The was with in The cells were with in for in and with serum in for The cells were in serum in containing a of for in with in containing a of for and in to cells were on with RCE cells were on with PBS, in for with for and with for The were with were on a with and The were with RCE in were in for and were stimulated with for the was and the cells were with of with was to the at of with and was and for at by addition of of with The were several and to The of individual was using a of the was by of was To determine which of the phorbol ester-sensitive PKC were in RCE cells, and membrane of quiescent and RCE cells were to and with to and as well as an isoenzyme not to phorbol these and a of were in either the or of RCE, to with human endothelial cells K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (69) Google Scholar); serum stimulation for did not expression of or but did a in PKCθ at the protein and the Thus, these results suggested the that of the of PKCθ might endothelial proliferation by serum or phorbol To the a constitutively mutation of PKCθ was with a PKCθ cDNA (22Baier G. Telford D. Giampa L. Coggeshall K.M. Baier-Bitterlich G. Isakov N. Altman A. J. Biol. Chem. 1993; 268: 4997-5004Abstract Full Text PDF PubMed Google Scholar, 23Chang J.D. Xu Y. Raychowdhury M.K. Ware J.A. J. Biol. Chem. 1993; 268: 14208-14214Abstract Full Text PDF PubMed Google Scholar) and into RCE, and of these cells were by resistance and by and In with the PKCθ which to either or PKCθ, cells with PKCθ or more protein than did the cells that PKCθ single at were with this In a with the the cells with the Flag PKCθ and were indicating the of cloned PKCθ Although was some expression of the in the the expression increased the cells were with of PKC activity in of RCE the PKCθ-kn cells, and constitutively kinase-active PKCθ revealed that the PKCθ mutant not did not PKC activity but also was to inhibit PKCθ activity in a dominant-negative in the containing constitutively active PKCθ, as well as PKCθ, a of the activity was of of the PKCθ using the Flag revealed of PKCθ in was the with protein some activity or inhibition was constitutively in the and was increased by experiments that the PKCθ-kn was in the of PKCθ in kinase activity of and of PKCθ-kn RCE RCE cells were with one of the or the as protein of the RCE cells was with either or the kinase was as The of the kinase activity the of individual with the in separate and the or of with of of the with with the PKCθ and of the protein from the protein by in for the were with to The were on the and by with PKCθ of RCE cells revealed in quiescent cells, PKCθ is largely in the and stimulation with serum or contact with other cells induces translocation from the to the cell surface and The constitutively active or were to translocate to the surface to that with in the of serum or contact Thus, the intracellular of the PKCθ-kn mutant and the form of PKCθ in To determine the effects of PKCθ inhibition on endothelial proliferation, quiescent and PKCθ-kn RCE cells were stimulated with containing serum and cell were for revealed that RCE proliferation was inhibited by In the of the growth of the PKCθ-kn cells was with that of the cells the of the growth results that overexpression of the PKCθ-kn markedly inhibited RCE that PKCθ is required for of capillary is that inhibition was with PKCθ-kn that the to the To that these from the of the on the substrates of the the PKCθ-kn cells with a cDNA to the RCE cell and to whether the inhibitory effects The cells at a than did the cells but than PKCθ-kn expression of did not a on RCE these results that with the for the substrates this and that PKCθ is required for normal RCE cell growth. To determine whether inhibition of PKCθ was with cell the of and PKCθ-kn RCE to a wound in a of cells of of the defect by the PKCθ-kn RCE cells was by of the required to the revealed of the wound in in the RCE cells and the cells, which did not In the PKCθ-kn RCE the wound more and did not closure In the experiments RCE cells were in the of serum that proliferation did not to of the The experiments were in the of and results were not To that this from inhibition of PKCθ, also the RCE cells, with the that this with the PKCθ-kn for substrates. The cells the wound than did the PKCθ-kn cells, not as as the cells, indicating that the of PKCθ-kn was Thus, these results that the PKCθ in events for wound one of which is endothelial whether PKCθ-kn also formation of capillary structures in an in vitro of angiogenesis in a containing either or a membrane When in a normal RCE and cells rings in and PKCθ-kn cells, in capillary rings When in a normal RCE and cells capillary structures and the of these tubules to increased of cells with The of structures of PKCθ-kn cells to than that of RCE and cells, with and in addition to inhibiting endothelial proliferation and migration, PKCθ-kn formation of capillary including rings and formation of RCE cells on were on a in and or with and for and C, PKCθ-kn The morphology of the by RCE cells either with or was to that in In the of whether the in RCE proliferation by PKCθ-kn to a in the RCE cell Cell cycle revealed that the PKCθ-kn RCE cells a at the G2/M phase of the cells in this with of cells and of the cells not at following addition of serum In to these the of required to as by the of an in the of cells in phase following from was not different in RCE cells, the cells and PKCθ-kn cells of in Thus, these results that the inhibition of cell proliferation in PKCθ-kn RCE is by a at the G2/M phase of the cell PKCθ is not required for and into the of the the PKCθ-kn RCE cells in morphology from either the RCE or RCE were or RCE cells and PKCθ-kn The cells a of capillary endothelial cells and were The PKCθ-kn cells revealed an and were in or of this and the that PKC activation been to in maintenance of the actin in endothelial and other cell 1993; PubMed Scopus (40) Google Scholar), whether of PKCθ activity formation of actin revealed that RCE prominent stress fibers of with of actin in a peripheral band containing filopodia were also in the cells C and as well as in the RCE cells, which also prominent actin stress In the PKCθ-kn RCE a prominent peripheral band of actin but of these stress fibers or filopodia in the actin largely as not into Cell ruffling the of RCE and the of PKCθ-kn cells was indicating that PKCθ is not a in this of In the cells which PKCθ-kn cells were with a and cell morphology the of those in the RCE cells, further that PKCθ is in the formation of stress fibers of the that receptors been to in angiogenesis P.C. S. R. D. Cheresh D.A. J. 1995; PubMed Scopus Google Scholar), the of PKCθ-kn expression on of RCE to of of the endothelial cells to was in the PKCθ-kn cells not that this PKC isoenzyme not participate in events that are to the of or other receptors to to an This study that PKCθ is a of the that to endothelial and formation of structures by were using a PKCθ-kn by methods by others I. L. J. J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, S. Y. Y. A. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, J.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) on other PKC to a and The of this is suggested by the inhibition of PKCθ activity in the in vitro the that changes in endothelial cell increased upon expression of the gene at following in the cells, that the observed from this the PKCθ-kn is for the substrates of PKCθ is suggested by the of the by overexpression of PKCθ in the affected Thus, it is not that other or also with these it seems that the observed results from inhibition of of PKCθ substrates. The of these to the of the intracellular that angiogenesis and repair of a vessel is by the in endothelial proliferation, wound and capillary and formation in the endothelial cells the PKCθ is in the and of RCE, indicating that matrix contact is not sufficient to translocation of the isoenzyme to the surface PKCθ to the cell upon either serum stimulation or cell endothelial cells and reenter the cell cycle as a to migration and proliferation contact inhibition is (3Coomber B.L. Gotlieb A.I. Arteriosclerosis. 1990; 10: 215-222Crossref PubMed Google Scholar), it seems that PKCθ, to the cell is stimulated by one or more of the in contact and on substrates to formation of actin stress fibers and activation of the PKC family by at least some mitogens or by phorbol esters been to and sufficient for endothelial proliferation and angiogenesis in vitro and in vivo (13Daviet I. Herbert J.M. Maffrand M. Biochem. Biophys. Res. Commun. 1989; 158: 584-589Crossref PubMed Scopus (18) Google Scholar, 14Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1227) Google Scholar, 15Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (69) Google Scholar). In others used different experimental and have that addition of phorbol esters endothelial cells in phase by inhibiting the of and also either or depending upon the phase of in which they are C. Sasaguri T. Ishida A. Ogata J. Am. J. Physiol. 1996; 270: C170-C178Crossref PubMed Google N. M. Y. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). This that individual of PKC, either or by intracellular might mediate these of the constitutively active PKCθ mutant does not inhibit cell cycle or RCE proliferation, that the inhibitory effects observed following phorbol esters not from stimulation of this the other the results with the PKCθ-kn cells that this individual PKC family is required for and formation of actin structures and is a for the of the by phorbol endothelial proliferation and angiogenesis from growth K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (69) Google Scholar) or vascular endothelial growth M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. 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