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Reentry into the Cell Cycle of Contact-inhibited Vascular Endothelial Cells by a Phosphatase Inhibitor

POSSIBLE INVOLVEMENT OF EXTRACELLULAR SIGNAL-REGULATED KINASE AND PHOSPHATIDYLINOSITOL 3-KINASE*
Open AccessPublished:February 04, 2000DOI:https://doi.org/10.1074/jbc.275.5.3637
      Vascular endothelial cells are unique in that they exit from the cell cycle when they come into contact with each other. Although the phenomenon is called “contact inhibition,” little is known about the cellular mechanisms involved. Here we show that the phosphatase inhibitor sodium orthovanadate (SOV) induced the reentry of contact-inhibited human umbilical vascular endothelial cells (HUVECs) into the cell cycle and that reentry was associated with activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K)/Akt pathways. SOV stimulated [3H]thymidine uptake of contact-inhibited HUVECs in a time- and dose-dependent manner. SOV-induced increase in [3H]thymidine uptake was significantly inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by the PI 3-K inhibitor LY294002. SOV also stimulated the expression of cyclin D1, cyclin E, and cyclin A, and the activity of CDK2 kinase, whereas it decreased the expression of p27 kip1. In marked contrast, growth media alone did not induce these changes. Furthermore, these SOV-induced changes were abolished by pretreatment with PD98059 and LY294002. SOV stimulated phosphorylation of ERK and Akt in contact-inhibited HUVECs, while growth media alone did not. This phosphorylation was associated with inhibition of phosphatase activity in the cells. Finally, overexpression of high cell density-enhanced protein tyrosine phosphatase 1 inhibited c-fos and cyclin A promoter activity. Taken together, our results suggest that in contact-inhibited HUVECs, increased phosphatase activity suppressed the ERK and PI 3-K/Akt pathways, resulting in exit from the cell cycle by down-regulation of cyclin D1, cyclin E, and cyclin A and by up-regulation of p27 kip1.
      EC
      endothelial cell
      HUVEC
      human umbilical vascular endothelial cell
      PTPase
      protein-tyrosine phosphatase
      SOV
      sodium orthovanadate
      DEP-1
      high cell density-enhanced PTPase 1
      CDK
      cyclin-dependent kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      ERK
      extracellular signal-regulated kinase
      BAEC
      bovine aortic endothelial cell
      PBS
      phosphate-buffered saline
      HA
      hemagglutinin
      PI 3-K
      phosphatidylinositol 3-kinase
      Vascular endothelial cells (ECs)1 play a variety of pathophysiological roles such as provision of a barrier through which substances are transported into vessel walls, maintenance of vascular tone by releasing vasoactive substances including nitric oxide and endothelin, and oxidization of lipoproteins (
      • Ross R.
      ). ECs are unique because they grow as a strict monolayer, and they exit from the cell cycle once they come into contact with each other. Although the phenomenon is well known and called “contact inhibition,” little is known about the molecular mechanisms involved.
      Several lines of evidence suggest that up-regulation of phosphatase activity may be implicated in density-dependent growth arrest. NRK-1 cells were transformed by treatment with the phosphatase inhibitor sodium orthovanadate (SOV), and transformation was accompanied by increases in protein phosphorylation in the cells (
      • Klarlund J.K.
      ). Protein-tyrosine phosphatase (PTPase) activity was increased in human umbilical vein endothelial cells (HUVECs) harvested at high density (
      • Gaits F.
      • Li R.Y.
      • Ragab A.
      • Ragab-Thomas J.M.
      • Chap H.
      ). The increase in PTPase activity was also observed in Swiss 3T3 fibroblasts whose growth was arrested at high density (
      • Pallen C.J.
      • Tong P.H.
      ). Recently, a novel class of receptor-like PTPases was isolated and named high cell density-enhanced PTPase 1 (DEP-1). DEP-1 has an extracellular domain that contains eight fibronectin type III motifs, a single transmembrane domain, and a single intracellular PTPase domain. The expression level and the PTPase activity of DEP-1 were increased in WI-38 and AG1518 cells harvested at high density (
      • Ostman A.
      • Yang Q.
      • Tonks N.K.
      ). Although these findings suggest that increased PTPase activity in the cells might counteract with protein phosphorylation, which leads to cell proliferation, little is known as to which intracellular signaling pathways are affected by the increase in PTPase activity and how the increased PTPase activity finally affects the cell cycle regulatory machinery.
      Cell cycle progression is regulated by serine/threonine kinases termed cyclin-dependent kinases (CDKs), the activities of which oscillate during the cell cycle. CDKs are associated with the positive coactivators cyclins and the negative regulators, CDK inhibitors (
      • Pines J.
      ,
      • Sherr C.J.
      • Roberts J.M.
      ). In mammalian cells, cyclin D-CDK4/CDK6, cyclin E-CDK2, cyclin A-CDK2, and cyclin B-Cdc2 are the main cyclin-CDK complexes that regulate the progression of G1, G1/S, S, and G2/M phases, respectively. CDK inhibitors comprise two families, the Ink4 and Cip/Kip families. CDK inhibitors of the Cip/Kip family are of particular interest in that they inhibit the activity of a broader spectrum of CDKs including CDK2, CDK4, and CDK6 (
      • Harper J.W.
      • Elledge S.J.
      • Keyomarsi K.
      • Dynlacht B.
      • Tsai L.H.
      • Zhang P.
      • Dobrowolski S.
      • Bai C.
      • Connell-Crowley L.
      • Swindell E.
      • Fox M.P.
      • Wei N.
      ). The Cip/Kip family is composed of p21 waf1/cip1, p27 kip1, and p57 kip2 (
      • El-Deiry W.S.
      • Tokino T.
      • Velculescu V.E.
      • Levy D.B.
      • Parsons R.
      • Trent J.M.
      • Lin D.
      • Mercer W.E.
      • Kinzler K.W.
      • Vogelstein B.
      ,
      • Harper J.W.
      • Adami G.R.
      • Wei N.
      • Keyomarsi K.
      • Elledge S.J.
      ,
      • Lee M.-H.
      • Reynisdottir I.
      • Massague J.
      ,
      • Polyak K.
      • Lee M.-H.
      • Erdjument-Bromage H.
      • Koff A.
      • Roberts J.M.
      • Tempst P.
      • Massague J.
      ). The expression level of p27 kip1 is reportedly increased in growth-arrested cells by contact inhibition or by stimulation with transforming growth factor-β (
      • Hengst L.
      • Dulic V.
      • Slingerland J.M.
      • Lees E.
      • Reed S.I.
      ,
      • Slingerland J.M.
      • Hengst L.
      • Pan C.H.
      • Alexander D.
      • Stampfer M.R.
      • Reed S.I.
      ,
      • Polyak K.
      • Kato J.
      • Solomon M.J.
      • Sherr C.J.
      • Massague J.
      • Roberts J.M.
      • Koff A.
      ,
      • Winston J.
      • Dong F.
      • Pledger W.J.
      ,
      • Kato A.
      • Takahashi H.
      • Takahashi Y.
      • Matsushime H.
      ).
      Recent evidence suggests that several intracellular signaling pathways are linked to the cell cycle regulatory machinery. Among them, the p21 ras (RAS)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is the most characterized pathway. It is reported that constitutively active MEK is sufficient to transform cells or induce differentiation (
      • Cowley S.
      • Paterson H.
      • Kemp P.
      • Marshall C.J.
      ,
      • Mansour S.J.
      • Matten W.T.
      • Hermann A.S.
      • Candia J.M.
      • Rong S.
      • Fukasawa K.
      • Vande Woude G.F.
      • Ahn N.G.
      ), suggesting that the MEK/ERK pathway is implicated in the cell cycle regulation. Several studies have indicated, using a dominant-negative Ras mutant, that the Ras signaling pathway is involved in the induction of cyclin D1 protein, CDK4 kinase activity, and CDK2 kinase activity and in the down-regulation of p27 kip1 (
      • Aktas H.
      • Cai H.
      • Cooper G.M.
      ,
      • Takuwa N.
      • Takuwa Y.
      ). Furthermore, the ERK pathway is reportedly implicated in vascular endothelial cell growth factor-induced EC proliferation by stimulating cyclin D1 synthesis and CDK4 kinase activity (
      • Pedram A.
      • Razandi M.
      • Levin E.R.
      ). Protein kinase C (PKC) stimulates cell proliferation or induces cell cycle arrest in vascular ECs, which appears to depend upon the timing of PKC activation during the cell cycle and PKC isozymes expressed in the cells (
      • Tang S.
      • Morgan K.G.
      • Parker C.
      • Ware J.A.
      ,
      • Harrington E.O.
      • Loffler J.
      • Nelson P.R.
      • Kent K.C.
      • Simons M.
      • Ware J.A.
      ,
      • Kosaka C.
      • Sasaguri T.
      • Ishida A.
      • Ogata J.
      ,
      • Zhou W.
      • Takuwa N.
      • Kumada M.
      • Takuwa Y.
      ). The PI 3-K-mediated pathways also seem to be implicated in cell cycle progression, because a retrovirus-encoded PI 3-K could transform fibroblasts (
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Roberts T.M.
      • Vogt P.K.
      ). However, it remains unclear as to how these intracellular signaling pathways are regulated in contact-inhibited vascular ECs.
      To investigate the cellular mechanisms for density-dependent growth arrest and to apply those mechanisms to the regulation of the growth of other cell types, such as vascular smooth muscle cells, we examined in the present study whether treatment with the phosphatase inhibitor SOV induced reentry of contact-inhibited vascular ECs into the cell cycle by examining [3H]thymidine incorporation, expression levels of cyclins, CDKs and CDK inhibitors, and CDK2 kinase activity. We also studied the effects of inhibition of the ERK-, PKC-, and PI 3-K-mediated pathways on SOV-induced cell cycle reentry. Finally, we examined the effect of overexpression of DEP-1 on c-fos and cyclin A promoter activity.

      DISCUSSION

      In the present study, we have shown that contact-inhibited HUVECs reentered the cell cycle in the presence of SOV by examining [3H]thymidine incorporation, protein expression levels of cyclins, CDKs, and CDK inhibitors, and CDK2 kinase activity. We have also demonstrated that SOV-induced cell cycle reentry of contact-inhibited HUVECs was associated with activation of ERK and Akt and that SOV-induced cell cycle reentry was inhibited by pretreatment with the MEK1/2 inhibitor PD98059 and the PI 3-K inhibitor LY294002. Furthermore, we have shown that overexpression of DEP-1 inhibited c-fos and cyclin A promoter activity in BAECs, which was restored by co-expression of the constitutively active Ras mutant RasG12V. A previous report has suggested that at an appropriate concentration, SOV could transform cells and that transformation was associated with increased protein phosphorylation in the cells (
      • Klarlund J.K.
      ). However, little is known as to which intracellular signaling pathways are activated by SOV and how treatment with SOV is finally linked to the cell cycle regulatory machinery. Our results showed down-regulation of cyclin D1, cyclin E, cyclin A, and CDK2 kinase activity and up-regulation of p27 kip1 in contact-inhibited HUVECs. Our results also demonstrated that treatment of contact-inhibited HUVECs with SOV finally induced an increased expression of cyclin D1, cyclin E, and cyclin A, an increased CDK2 kinase activity, and down-regulation of p27 kip1, all of which could potentially stimulate cells to reenter the cell cycle. Recent reports suggested that up-regulation of p27 kip1 was associated with density-dependent growth arrest or growth arrest induced by transforming growth factor-β (
      • Hengst L.
      • Dulic V.
      • Slingerland J.M.
      • Lees E.
      • Reed S.I.
      ,
      • Slingerland J.M.
      • Hengst L.
      • Pan C.H.
      • Alexander D.
      • Stampfer M.R.
      • Reed S.I.
      ,
      • Polyak K.
      • Kato J.
      • Solomon M.J.
      • Sherr C.J.
      • Massague J.
      • Roberts J.M.
      • Koff A.
      ,
      • Winston J.
      • Dong F.
      • Pledger W.J.
      ,
      • Kato A.
      • Takahashi H.
      • Takahashi Y.
      • Matsushime H.
      ). However, it is also reported that p27 kip1 (−,−) cells were growth-arrested by contact inhibition (
      • Nakayama K.
      • Ishida N.
      • Shirane M.
      • Inomata A.
      • Inoue T.
      • Shishido N.
      • Horii I.
      • Loh D.Y.
      • Nakayama K.
      ), suggesting that up-regulation of p27 kip1 was not the sole factor that induced growth arrest. In this regard, it is of interest to note that the transcript level and promoter activity of cyclin A were reduced in contact-inhibited BAECs (
      • Yoshizumi M.
      • Hsieh C.M.
      • Zhou F.
      • Tsai J.C.
      • Patterson C.
      • Perrella M.A.
      • Lee M.E.
      ). Thus, it is possible that the down-regulation of cyclin D1, cyclin E, and cyclin A contributes to the growth arrest by contact inhibition in HUVECs. Although p21 cip1 expression level was also increased by SOV, this was transient. It has been reported that p21 cip1 expression was transiently increased by mitogens (
      • Olson M.F.
      • Paterson H.F.
      • Marshall C.J.
      ). Our results are compatible with those of that report.
      Our results indicated that inhibition of the MEK- and PI 3-K-mediated pathways resulted in suppression of SOV-induced up-regulation of cyclin D1, cyclin E, cyclin A, and CDK2 kinase activity and restoration of SOV-induced down-regulation of p27 kip1. Several lines of evidence have suggested that the Ras/MEK/ERK- and PI 3-K-mediated pathways are linked to the cell cycle regulatory machinery. A constitutively active MEK could transform cells or induce differentiation, which depended upon cell types (
      • Cowley S.
      • Paterson H.
      • Kemp P.
      • Marshall C.J.
      ,
      • Mansour S.J.
      • Matten W.T.
      • Hermann A.S.
      • Candia J.M.
      • Rong S.
      • Fukasawa K.
      • Vande Woude G.F.
      • Ahn N.G.
      ). In several studies, it was demonstrated, using a dominant negative Ras mutant, that Ras signaling pathways were involved in the up-regulation of cyclin D1, cyclin A, and CDK2 kinase activity and in down-regulation of p27 kip1 (
      • Aktas H.
      • Cai H.
      • Cooper G.M.
      ,
      • Takuwa N.
      • Takuwa Y.
      ). Involvement of MEK/ERK in cyclin D1 up-regulation was also suggested in a report in which PD98059 was used to inhibit platelet-derived growth factor-induced ERK activation (
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ). PI 3-K also appears to be implicated in cell cycle progression. A retrovirus-encoded PI 3-K could transform fibroblasts (
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Roberts T.M.
      • Vogt P.K.
      ). PI 3-K may induce cell cycle progression via activation of p70S6K, which seems to play roles in the initiation of protein synthesis (
      • Proud C.G.
      ,
      • Erikson R.L.
      ). A recent report showed a direct link between Akt activation and stabilization of cyclin D1. In that report, glycogen synthase kinase-3β phosphorylated cyclin D1 on Thr286, which stimulated the degradation of cyclin D1. PI 3-K/Akt phosphorylated and inactivated glycogen synthase kinase-3β, resulting in the stabilization of cyclin D1 (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ). Furthermore, it was suggested that the PI 3-K-mediated pathway was implicated in p27 kip1 down-regulation by mitogens, because LY294002 and wortmannin restored p27 kip1 expression (
      • Takuwa N.
      • Takuwa Y.
      ,
      • St. Croix B.
      • Sheehan C.
      • Rak J.W.
      • Florenes V.A.
      • Slingerland J.M.
      • Kerbel R.S.
      ). Thus, our results were basically compatible with those of previous reports. Although PKC inhibition did not significantly affect the SOV-induced cell cycle reentry in our system, the involvement of PKC could not be excluded, because we did not examine which classes of PKC were indeed inhibited by calphostin C or GF109203X.
      Our results suggested that the MEK/ERK and PI 3-K/Akt pathways were shut down in contact-inhibited HUVECs and that these pathways were reactivated in the presence of SOV. Furthermore, the reactivation of the MEK/ERK and PI 3-K/Akt pathways by SOV treatment correlated with the suppression of SOV-sensitive phosphatases, suggesting that SOV-sensitive phosphatases might be involved in the down-regulation of those pathways, although it was not clear whether the SOV-sensitive phosphatases inhibited phosphorylation of molecules such as Shc, which leads to the activation of the MEK/ERK and PI 3-K/Akt pathways, or stimulated dephosphorylation of ERK and Akt or both. It should be noted that the phosphorylation of ERK was reportedly decreased in contact-inhibited vascular endothelial cells, while activation of Ras and MEK was not impaired (
      • Vinals F.
      • Pouyssegur J.
      ). The results indicate that dephosphorylation of ERK was stimulated and that the pathways located upstream of ERK were intact in contact-inhibited vascular endothelial cells. Recent reports have suggested that PKC-ζ, a PKC isozyme that belongs to a class of atypical PKC, directly phosphorylates and activates MEK, resulting in activation of ERK (
      • Diaz-Meco M.T.
      • Dominguez I.
      • Sanz L.
      • Dent P.
      • Lozano J.
      • Municio M.M.
      • Berra E.
      • Hay R.T.
      • Sturgill T.W.
      • Moscat J.
      ,
      • Schonwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ). It has also been reported that PKC-ζ is a downstream target of PI 3-K (
      • Standaert M.L.
      • Galloway L.
      • Karnam P.
      • Bandyopadhyay G.
      • Moscat J.
      • Farese R.V.
      ,
      • Takeda H.
      • Matozaki T.
      • Takada T.
      • Noguchi T.
      • Yamao T.
      • Tsuda M.
      • Ochi F.
      • Fukunaga K.
      • Inagaki K.
      • Kasuga M.
      ). We therefore examined whether LY294002 inhibited ERK phosphorylation via inhibition of PI 3-K/PKC-ζ. Our results, however, suggested that ERK phosphorylation occurred in the presence of LY294002, indicating that the involvement of PKC-ζ was not the major pathway of activation of the ERK pathway by SOV. Our results also showed that PD98059 did not inhibit Akt phosphorylation, suggesting that activation of either the ERK pathway alone or the PI 3-K/Akt pathway alone with the inhibition of the other pathway was not sufficient to induce cell cycle reentry in contact-inhibited HUVECs.
      To study the role of a specific phosphatase in vascular ECs, we examined the effects of DEP-1 overexpression on c-fos and cyclin A promoter activity in vascular ECs. Our results suggest that overexpression of DEP-1 could potentially inhibit cell cycle progression in vascular ECs. These results were compatible with those of a previous study in which growth of breast cancer cells was inhibited by overexpressed DEP-1 (
      • Keane M.M.
      • Lowrey G.A.
      • Ettenberg S.A.
      • Dayton M.A.
      • Lipkowitz S.
      ). Although we did not exclude the possibility that RasG12V inhibited DEP-1 activity, it is possible that the inhibitory effect of DEP-1 on cell cycle progression was mediated by, at least in part, suppression of the Ras-dependent pathways such as ERK and PI 3-K, because RasG12V restored the activity of the cyclin A promoter. Our results showed that the inhibitory effect of DEP-1 on cyclin A promoter activity was weaker than that of RasS17N. However, this did not mean that the role of phosphatases in density-dependent growth arrest was small, because DEP-1 was not the only phosphatase whose activity was increased in density-dependent growth arrest (
      • Gaits F.
      • Li R.Y.
      • Ragab A.
      • Ragab-Thomas J.M.
      • Chap H.
      ,
      • Pallen C.J.
      • Tong P.H.
      ).
      Taken together, our results and those of other authors (
      • Gaits F.
      • Li R.Y.
      • Ragab A.
      • Ragab-Thomas J.M.
      • Chap H.
      ,
      • Pallen C.J.
      • Tong P.H.
      ,
      • Ostman A.
      • Yang Q.
      • Tonks N.K.
      ,
      • Vinals F.
      • Pouyssegur J.
      ) suggest the following scenario. The increased phosphatase activities in contact-inhibited vascular ECs cause down-regulation of the MEK/ERK and PI 3-K/Akt pathways, which in turn leads to the exit from the cell cycle due to down-regulation of cyclin D1, cyclin E, and cyclin A expression, and CDK2 kinase activity, and up-regulation of p27 kip1 expression. However, these changes are reversible, and once the phosphatases are inhibited, contact-inhibited vascular ECs reenter the cell cycle. Thus, phosphatases, especially those whose activities are increased in density-dependent growth arrest, can be used to control the growth of other cell types, such as vascular smooth muscle cells. Further studies are required to identify other specific phosphatases that are implicated in density-dependent growth arrest and to elucidate the mechanisms by which the MEK/ERK and PI 3-K/Akt pathways are down-regulated by phosphatases.

      Acknowledgments

      We thank Dr. Tadashi Yamamoto for kindly providing the PME18S-mouse DEP-1 plasmid. We also thank Etsuko Taira and Marie Morita for technical assistance.

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