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W2080646585 abstract "The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation. The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation. 5′-terminal oligopyrimidine S6 kinase mitogen-activated protein kinase extracellular signal-regulated kinase epidermal growth factor 5-bromo-2′-deoxyuridine embryonic day 19 During the last 3 days of gestation in the rat, fetal weight more than triples, with liver weight increasing proportionately (1Mayor F. Cuezva J.M. Biol. Neonate. 1985; 48: 185-196Crossref PubMed Scopus (62) Google Scholar). This rate of hepatic growth slows markedly at term; and although there is a restoration of vigorous growth in the neonatal period, the extraordinary rate of growth seen in late gestation is never again attained. We have shown previously that late fetal and neonatal development is associated with a distinctive pattern of hepatocyte proliferation during the perinatal period (2Curran T.R.J. Bahner R.I.J. Oh W. Gruppuso P.A. Exp. Cell Res. 1993; 209: 53-57Crossref PubMed Scopus (49) Google Scholar, 3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar). Our work has focused on the developmentally regulated signal transduction mechanisms that control hepatocyte proliferation during late gestation and beyond.Activation of hepatocyte proliferation following partial hepatectomy in the adult rat has been shown to correlate with the intracellular activation of several interacting protein kinase cascades (Reviewed in Ref. 4Diehl A.M. Rai R.M. FASEB J. 1996; 10: 215-227Crossref PubMed Scopus (213) Google Scholar). One of the signaling cascades that is activated after partial hepatectomy leads to the phosphorylation of ribosomal protein S6 (5Nemenoff R.A. Price D.J. Mendelsohn M.J. Carter E.A. Avruch J. J. Biol. Chem. 1988; 263: 19455-19460Abstract Full Text PDF PubMed Google Scholar) and is characterized by its sensitivity to the immunosuppressant rapamycin (6Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Studies have established that the target of rapamycin is mTOR (mammalian target ofrapamycin; also named FRAP/RAFT1) (7Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). Rapamycin forms a complex with the immunophilin peptidylprolyl isomerase FKBP12, which binds to mTOR and inhibits its ability to phosphorylate substrates such as S6 kinase and 4E-BP1. Phosphorylation of ribosomal protein S6, located in the 40 S subunit, is thought to be required for the translation of a subset of mRNAs that contain a 5′-oligopyrimidine tract at their transcriptional start sites (5′-terminal oligopyrimidine (5′-TOP)1 mRNAs) (8Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar). 5′-TOP mRNAs may number as few as 100–200, but they can account for 20–30% of total cellular mRNA. They encode many of the components of the translational apparatus, including ribosomal proteins and elongation factors that are necessary for cell cycle progression. Recently, Volarevic et al. (9Volarevic S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (314) Google Scholar) developed a system for the conditional deletion of protein S6 in mouse liver. They made the unexpected observation that loss of S6 had no effect on hepatocyte growth in response to refeeding after a fast, but that hepatocyte proliferation in response to partial hepatectomy was completely abolished. This led these authors to conclude that abrogation of 40 S ribosome biogenesis may induce checkpoint control that prevents cell cycle progression.Thomas and co-workers (10Jeno P. Ballou L.M. Novak-Hofer I. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 406-410Crossref PubMed Scopus (144) Google Scholar) first purified and characterized the kinase activity responsible for S6 phosphorylation in mitogen-stimulated Swiss mouse 3T3 cells more than 10 years ago. Subsequent purification and cloning showed that two isoforms of this original kinase are produced from the same transcript (reviewed by Dufner and Thomas (8Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar)). Null mutation in mice for the p70/p85 S6 kinases, which we will refer to as S6K1, is associated with a proportional 20% decrease in somatic growth (11Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). Mouse embryo fibroblasts derived from these animals showed diminished (but not absent) S6 phosphorylation, leading to the discovery of another physiological S6 kinase. This enzyme, a mitogen-responsive S6K1 homolog designated S6K2, has since been identified by other laboratories (12Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar). It has recently been determined that S6K1 and S6K2 are regulated similarly by effectors of the phosphatidylinositol 3-kinase pathway, including Cdc42, Rac, protein kinase Cζ, and phospholipid-dependent kinase-1 (14Martin K.A. Schalm S.S. Richardson C. Romanelli A. Keon K.L. Blenis J. J. Biol. Chem. 2001; 276: 7884-7891Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Our prior studies on mitogenic signaling during liver development in the rat have suggested that the well characterized pathways that mediate growth factor-induced mitogenesis in adult rat hepatocytes are not operative in the late gestation fetus. Our results have indicated an alternative means for the up-regulation of c-myc via RNA stabilization (15Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar), uncoupling of the prototypical MAPK pathway that terminates in ERK1/2 (16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and post-transcriptional induction of cyclin D1 (18Awad M.M. Gruppuso P.A. Cell Growth Differ. 2000; 11: 325-334PubMed Google Scholar). Given the established role of S6 phosphorylation in hepatocyte proliferation, we undertook a study of the hepatic signal transduction pathways terminating in ribosomal protein S6 phosphorylation in fetal and adult rats.DISCUSSIONFor a cell to proliferate, it must up-regulate the biosynthetic apparatus needed to support cell growth. Studies have shown that the mRNA transcripts for all ribosomal proteins and protein synthesis elongation factors contain an unusual oligopyrimidine tract at their transcriptional start sites, commonly referred to as a 5′-TOP. The translation of these mRNAs has been shown to be dependent on S6K1 activation, presumably mediated by an increase in S6 phosphorylation (28Jefferies H.B. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (550) Google Scholar). Studies have demonstrated that 5′-TOP mRNA translation is selectively inhibited by the immunosuppressant rapamycin, a potent inhibitor of S6 kinase activity (29Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar).Our initial studies led to the unexpected finding that basal hepatic S6K1 activity in the late gestation fetal rat is lower than in adult animals. Given our prior observations demonstrating that late gestation fetal rat hepatocytes proliferate at a high rate both in vivo and in vitro (2Curran T.R.J. Bahner R.I.J. Oh W. Gruppuso P.A. Exp. Cell Res. 1993; 209: 53-57Crossref PubMed Scopus (49) Google Scholar, 3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar), the low basal S6K1 activity represents a dissociation between cell proliferation and activity of this key signaling enzyme (3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar). Furthermore, S6K1 activation in response to the in vivo administration of insulin or EGF was markedly attenuated in fetal liver compared with adult liver. This is reminiscent of our observations showing similar uncoupling of the ERK MAPK pathway in late gestation liver (16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We proceeded to demonstrate that, despite the low basal activity of S6K1, ribosomal protein S6 is indeed hyperphosphorylated in fetal liver. We considered S6K2 to be a candidate to account for the phosphorylation of S6 in fetal liver. Basal S6K2 activity was ∼2-fold higher in E19 liver than in adult liver, consistent with a role for S6K2 in maintaining S6 phosphorylation in vivo. However, the relatively low S6K2specific activity that we measured is entirely consistent with the possibility of an alternative mechanism for S6 hyperphosphorylation in fetal liver. One such explanation would be the existence of an alternative S6 kinase. Another possibility is a lower level of S6 phosphatase activity in fetal liver ribosomes. Studies by Olivieret al. (22Olivier A.R. Ballou L.M. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4720-4724Crossref PubMed Scopus (45) Google Scholar) indicate that the catalytic subunit of protein phosphatase-1 mediates the dephosphorylation of ribosomal protein S6. Low protein phosphatase-1 expression, decreased localization to ribosomes through decreased expression of associated subunits, or inhibition of ribosomal protein phosphatase-1 activity could serve to maintain S6 in a hyperphosphorylated state, even in the absence of excess kinase activity in fetal liver.To dissect the pathways accounting for the fetal liver S6 hyperphosphorylation, we administered rapamycin to intact animals. These studies were undertaken to test the hypothesis that fetal hepatocytes utilize an alternative, rapamycin-resistant pathway for S6 phosphorylation. Indeed, we found that rapamycin administration to E19 rats in situ did not inhibit DNA synthesis despite potent inhibition of S6K1 and S6K2. This led to analysis for the sensitivity of S6 phosphorylation to rapamycin and the unexpected finding that fetal hepatocyte proliferation in vivo could proceed following rapamycin administration even though S6 phosphorylation was markedly decreased. These results contrasted with those in adult animals. We employed a commonly used method for inducing adult hepatocytes to enter the cell cycle, a two-thirds hepatectomy (30Fausto N. Webber E.M. Arias A.M.B.J. Fausto N. Jacoby W.B. Schlacter D. Shafrits D.A. The Liver: Biology and Pathobiology. Raven Press, Ltd., New York1994: 1059-1084Google Scholar). This resulted in an increase in S6K1 activity as well as a marked increase in S6 phosphorylation. As anticipated, rapamycin effectively blocked in vivo S6 phosphorylation and hepatocyte DNA synthesis following partial hepatectomy. These results are consistent with the recently published report by Jiang et al. (25Jiang Y.-P. Ballou L.M. Lin R.Z. J. Biol. Chem. 2001; 276: 10943-10951Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) showing that rapamycin blocks activation of p70S6K in a dose-dependent manner and inhibits recovery of liver mass after a partial hepatectomy.There are precedents for the dissociation of S6 phosphorylation and cell proliferation. Kawasome et al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) performed studies that point to the existence of S6-independent mechanisms for 5′-TOP mRNA translation. Their S6K1 knockout murine embryonic stem cells did not show any S6 phosphorylation in vivo. However, 40% of total cellular eukaryotic elongation factor-1α mRNA, a 5′-TOP mRNA, was detected in polysomal fractions, indicating active eukaryotic elongation factor-1α translation. Prior to these studies, S6K1 activation was generally thought to be essential for G1/S transition. This was based on the work of Laneet al. (32Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar), who showed that microinjection of antibodies against S6K1 prevented the entry of cells into the cell cycle. Kawasomeet al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) found that the S6K1 null embryonic stem cells proliferated well, albeit slower than parental cells, indicating that S6K1 is not essential for G1/S transition. Interestingly, rapamycin inhibited the proliferation of the S6K1 null cells to the same extent as the parental cells, indicating that events independent of S6K1 activation, but probably requiring mTOR activation, are critical for cell proliferation.This uncoupling of S6K1 activity and proliferation had previously been shown in activated T cells that were already cycling (33Terada N. Franklin R.A. Lucas J.J. Blenis J. Gelfand E.W. J. Biol. Chem. 1993; 268: 12062-12068Abstract Full Text PDF PubMed Google Scholar) as well as in erythroleukemic cell lines (34Calvo V. Wood M. Gjertson C. Vik T. Bierer B.E. Eur. J. Immunol. 1994; 24: 2664-2671Crossref PubMed Scopus (43) Google Scholar). Slavik et al. (35Slavik J.M. Lim D.-G. Burakoff S.J. Hafler D.A. J. Immunol. 2001; 166: 3201-3209Crossref PubMed Scopus (42) Google Scholar) recently found that p70S6K activation can be uncoupled from proliferation in both freshly isolated CD8+ T cells and CD8+ T cell clones, demonstrating for the first time rapamycin resistance in resting, nontransformed T cells.In studies by Withers et al. (36Withers D.J. Seufferlein T. Mann D. Garcia B. Jones N. Rozengurt E. J. Biol. Chem. 1997; 272: 2509-2514Crossref PubMed Scopus (42) Google Scholar) employing Swiss 3T3 cells, p70S6K activation was dissociated from DNA synthesis. Although rapamycin could inhibit both bombesin-mediated p70S6K activation and [3H]thymidine incorporation, the combination of bombesin plus insulin was able to stimulate DNA synthesis and cell cycle progression in the presence of rapamycin despite inhibition of p70S6K activity. These results were interpreted as indicating the presence of a rapamycin-insensitive mitogenic pathway in Swiss 3T3 cells.Another conclusion from our studies is that the signaling phenotype of fetal hepatocytes is altered when the cells are removed from theirin vivo milieu. In vivo, E19 hepatocytes showed a resistance to the S6K-activating effects of insulin and EGF and to the inhibitory effect of rapamycin on DNA synthesis. In contrast, primary cultures of E19 hepatocytes showed an intact response to hormonal activation of S6K1 and S6K2 as well as heightened sensitivity to the growth inhibitory effects of rapamycin. A similar in vivo toin vitro change in mitogenic signaling was seen in our studies on the developmental regulation of hepatic ERK1 and ERK2 (17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We have not established a mechanism for the uncoupling of proximal signaling from S6 kinase activation in developing liver. However, recent studies indicate a similar uncoupling of Akt activation and low levels of key proximal signaling molecules, including insulin receptor substrate-1 and Gab2. 2J. M. Boylan and P. A. Gruppuso, unpublished observations.As was the case for signaling to S6 kinases, fetal hepatocyte rapamycin resistance as measured by DNA synthesis was lost when hepatocytes were isolated and cultured. We observed an IC50 for both fetal and adult cells that was near 20 nm. The dose of rapamycin that we chose for our in vivo experiments was designed to achieve micromolar concentrations. Studies on “rapamycin-resistant” cancer cell lines have shown an IC50 in the micromolar range (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar).In summary, our results point to an alternative, S6 phosphorylation-independent pathway that supports fetal hepatocyte proliferation during late gestation. Our data indicate that phosphorylation of ribosomal protein S6 is not necessary for fetal hepatocyte proliferation in vivo. Given that translation of 5′-TOP mRNAs is thought to be essential for cellular proliferation, we conclude that fetal hepatocytes possess an alternative pathway for up-regulation of the translation of these genes. We found no evidence that this pathway is operative in adult hepatocytes. This conclusion is consistent with our previous studies (15Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar, 16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in that it suggests that mechanisms that mediate the proliferation of late gestation fetal hepatocytes are distinct from those that mediate the proliferation of mature hepatocytes in vivo. Finally, the rapamycin resistance displayed by a spectrum of cancer cells (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar) suggests that the mechanism for phospho-S6-independent hepatocyte proliferation in the late gestation fetal rat may prove relevant to liver carcinogenesis. During the last 3 days of gestation in the rat, fetal weight more than triples, with liver weight increasing proportionately (1Mayor F. Cuezva J.M. Biol. Neonate. 1985; 48: 185-196Crossref PubMed Scopus (62) Google Scholar). This rate of hepatic growth slows markedly at term; and although there is a restoration of vigorous growth in the neonatal period, the extraordinary rate of growth seen in late gestation is never again attained. We have shown previously that late fetal and neonatal development is associated with a distinctive pattern of hepatocyte proliferation during the perinatal period (2Curran T.R.J. Bahner R.I.J. Oh W. Gruppuso P.A. Exp. Cell Res. 1993; 209: 53-57Crossref PubMed Scopus (49) Google Scholar, 3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar). Our work has focused on the developmentally regulated signal transduction mechanisms that control hepatocyte proliferation during late gestation and beyond. Activation of hepatocyte proliferation following partial hepatectomy in the adult rat has been shown to correlate with the intracellular activation of several interacting protein kinase cascades (Reviewed in Ref. 4Diehl A.M. Rai R.M. FASEB J. 1996; 10: 215-227Crossref PubMed Scopus (213) Google Scholar). One of the signaling cascades that is activated after partial hepatectomy leads to the phosphorylation of ribosomal protein S6 (5Nemenoff R.A. Price D.J. Mendelsohn M.J. Carter E.A. Avruch J. J. Biol. Chem. 1988; 263: 19455-19460Abstract Full Text PDF PubMed Google Scholar) and is characterized by its sensitivity to the immunosuppressant rapamycin (6Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Studies have established that the target of rapamycin is mTOR (mammalian target ofrapamycin; also named FRAP/RAFT1) (7Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). Rapamycin forms a complex with the immunophilin peptidylprolyl isomerase FKBP12, which binds to mTOR and inhibits its ability to phosphorylate substrates such as S6 kinase and 4E-BP1. Phosphorylation of ribosomal protein S6, located in the 40 S subunit, is thought to be required for the translation of a subset of mRNAs that contain a 5′-oligopyrimidine tract at their transcriptional start sites (5′-terminal oligopyrimidine (5′-TOP)1 mRNAs) (8Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar). 5′-TOP mRNAs may number as few as 100–200, but they can account for 20–30% of total cellular mRNA. They encode many of the components of the translational apparatus, including ribosomal proteins and elongation factors that are necessary for cell cycle progression. Recently, Volarevic et al. (9Volarevic S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (314) Google Scholar) developed a system for the conditional deletion of protein S6 in mouse liver. They made the unexpected observation that loss of S6 had no effect on hepatocyte growth in response to refeeding after a fast, but that hepatocyte proliferation in response to partial hepatectomy was completely abolished. This led these authors to conclude that abrogation of 40 S ribosome biogenesis may induce checkpoint control that prevents cell cycle progression. Thomas and co-workers (10Jeno P. Ballou L.M. Novak-Hofer I. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 406-410Crossref PubMed Scopus (144) Google Scholar) first purified and characterized the kinase activity responsible for S6 phosphorylation in mitogen-stimulated Swiss mouse 3T3 cells more than 10 years ago. Subsequent purification and cloning showed that two isoforms of this original kinase are produced from the same transcript (reviewed by Dufner and Thomas (8Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar)). Null mutation in mice for the p70/p85 S6 kinases, which we will refer to as S6K1, is associated with a proportional 20% decrease in somatic growth (11Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). Mouse embryo fibroblasts derived from these animals showed diminished (but not absent) S6 phosphorylation, leading to the discovery of another physiological S6 kinase. This enzyme, a mitogen-responsive S6K1 homolog designated S6K2, has since been identified by other laboratories (12Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar). It has recently been determined that S6K1 and S6K2 are regulated similarly by effectors of the phosphatidylinositol 3-kinase pathway, including Cdc42, Rac, protein kinase Cζ, and phospholipid-dependent kinase-1 (14Martin K.A. Schalm S.S. Richardson C. Romanelli A. Keon K.L. Blenis J. J. Biol. Chem. 2001; 276: 7884-7891Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Our prior studies on mitogenic signaling during liver development in the rat have suggested that the well characterized pathways that mediate growth factor-induced mitogenesis in adult rat hepatocytes are not operative in the late gestation fetus. Our results have indicated an alternative means for the up-regulation of c-myc via RNA stabilization (15Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar), uncoupling of the prototypical MAPK pathway that terminates in ERK1/2 (16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and post-transcriptional induction of cyclin D1 (18Awad M.M. Gruppuso P.A. Cell Growth Differ. 2000; 11: 325-334PubMed Google Scholar). Given the established role of S6 phosphorylation in hepatocyte proliferation, we undertook a study of the hepatic signal transduction pathways terminating in ribosomal protein S6 phosphorylation in fetal and adult rats. DISCUSSIONFor a cell to proliferate, it must up-regulate the biosynthetic apparatus needed to support cell growth. Studies have shown that the mRNA transcripts for all ribosomal proteins and protein synthesis elongation factors contain an unusual oligopyrimidine tract at their transcriptional start sites, commonly referred to as a 5′-TOP. The translation of these mRNAs has been shown to be dependent on S6K1 activation, presumably mediated by an increase in S6 phosphorylation (28Jefferies H.B. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (550) Google Scholar). Studies have demonstrated that 5′-TOP mRNA translation is selectively inhibited by the immunosuppressant rapamycin, a potent inhibitor of S6 kinase activity (29Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar).Our initial studies led to the unexpected finding that basal hepatic S6K1 activity in the late gestation fetal rat is lower than in adult animals. Given our prior observations demonstrating that late gestation fetal rat hepatocytes proliferate at a high rate both in vivo and in vitro (2Curran T.R.J. Bahner R.I.J. Oh W. Gruppuso P.A. Exp. Cell Res. 1993; 209: 53-57Crossref PubMed Scopus (49) Google Scholar, 3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar), the low basal S6K1 activity represents a dissociation between cell proliferation and activity of this key signaling enzyme (3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar). Furthermore, S6K1 activation in response to the in vivo administration of insulin or EGF was markedly attenuated in fetal liver compared with adult liver. This is reminiscent of our observations showing similar uncoupling of the ERK MAPK pathway in late gestation liver (16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We proceeded to demonstrate that, despite the low basal activity of S6K1, ribosomal protein S6 is indeed hyperphosphorylated in fetal liver. We considered S6K2 to be a candidate to account for the phosphorylation of S6 in fetal liver. Basal S6K2 activity was ∼2-fold higher in E19 liver than in adult liver, consistent with a role for S6K2 in maintaining S6 phosphorylation in vivo. However, the relatively low S6K2specific activity that we measured is entirely consistent with the possibility of an alternative mechanism for S6 hyperphosphorylation in fetal liver. One such explanation would be the existence of an alternative S6 kinase. Another possibility is a lower level of S6 phosphatase activity in fetal liver ribosomes. Studies by Olivieret al. (22Olivier A.R. Ballou L.M. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4720-4724Crossref PubMed Scopus (45) Google Scholar) indicate that the catalytic subunit of protein phosphatase-1 mediates the dephosphorylation of ribosomal protein S6. Low protein phosphatase-1 expression, decreased localization to ribosomes through decreased expression of associated subunits, or inhibition of ribosomal protein phosphatase-1 activity could serve to maintain S6 in a hyperphosphorylated state, even in the absence of excess kinase activity in fetal liver.To dissect the pathways accounting for the fetal liver S6 hyperphosphorylation, we administered rapamycin to intact animals. These studies were undertaken to test the hypothesis that fetal hepatocytes utilize an alternative, rapamycin-resistant pathway for S6 phosphorylation. Indeed, we found that rapamycin administration to E19 rats in situ did not inhibit DNA synthesis despite potent inhibition of S6K1 and S6K2. This led to analysis for the sensitivity of S6 phosphorylation to rapamycin and the unexpected finding that fetal hepatocyte proliferation in vivo could proceed following rapamycin administration even though S6 phosphorylation was markedly decreased. These results contrasted with those in adult animals. We employed a commonly used method for inducing adult hepatocytes to enter the cell cycle, a two-thirds hepatectomy (30Fausto N. Webber E.M. Arias A.M.B.J. Fausto N. Jacoby W.B. Schlacter D. Shafrits D.A. The Liver: Biology and Pathobiology. Raven Press, Ltd., New York1994: 1059-1084Google Scholar). This resulted in an increase in S6K1 activity as well as a marked increase in S6 phosphorylation. As anticipated, rapamycin effectively blocked in vivo S6 phosphorylation and hepatocyte DNA synthesis following partial hepatectomy. These results are consistent with the recently published report by Jiang et al. (25Jiang Y.-P. Ballou L.M. Lin R.Z. J. Biol. Chem. 2001; 276: 10943-10951Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) showing that rapamycin blocks activation of p70S6K in a dose-dependent manner and inhibits recovery of liver mass after a partial hepatectomy.There are precedents for the dissociation of S6 phosphorylation and cell proliferation. Kawasome et al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) performed studies that point to the existence of S6-independent mechanisms for 5′-TOP mRNA translation. Their S6K1 knockout murine embryonic stem cells did not show any S6 phosphorylation in vivo. However, 40% of total cellular eukaryotic elongation factor-1α mRNA, a 5′-TOP mRNA, was detected in polysomal fractions, indicating active eukaryotic elongation factor-1α translation. Prior to these studies, S6K1 activation was generally thought to be essential for G1/S transition. This was based on the work of Laneet al. (32Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar), who showed that microinjection of antibodies against S6K1 prevented the entry of cells into the cell cycle. Kawasomeet al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) found that the S6K1 null embryonic stem cells proliferated well, albeit slower than parental cells, indicating that S6K1 is not essential for G1/S transition. Interestingly, rapamycin inhibited the proliferation of the S6K1 null cells to the same extent as the parental cells, indicating that events independent of S6K1 activation, but probably requiring mTOR activation, are critical for cell proliferation.This uncoupling of S6K1 activity and proliferation had previously been shown in activated T cells that were already cycling (33Terada N. Franklin R.A. Lucas J.J. Blenis J. Gelfand E.W. J. Biol. Chem. 1993; 268: 12062-12068Abstract Full Text PDF PubMed Google Scholar) as well as in erythroleukemic cell lines (34Calvo V. Wood M. Gjertson C. Vik T. Bierer B.E. Eur. J. Immunol. 1994; 24: 2664-2671Crossref PubMed Scopus (43) Google Scholar). Slavik et al. (35Slavik J.M. Lim D.-G. Burakoff S.J. Hafler D.A. J. Immunol. 2001; 166: 3201-3209Crossref PubMed Scopus (42) Google Scholar) recently found that p70S6K activation can be uncoupled from proliferation in both freshly isolated CD8+ T cells and CD8+ T cell clones, demonstrating for the first time rapamycin resistance in resting, nontransformed T cells.In studies by Withers et al. (36Withers D.J. Seufferlein T. Mann D. Garcia B. Jones N. Rozengurt E. J. Biol. Chem. 1997; 272: 2509-2514Crossref PubMed Scopus (42) Google Scholar) employing Swiss 3T3 cells, p70S6K activation was dissociated from DNA synthesis. Although rapamycin could inhibit both bombesin-mediated p70S6K activation and [3H]thymidine incorporation, the combination of bombesin plus insulin was able to stimulate DNA synthesis and cell cycle progression in the presence of rapamycin despite inhibition of p70S6K activity. These results were interpreted as indicating the presence of a rapamycin-insensitive mitogenic pathway in Swiss 3T3 cells.Another conclusion from our studies is that the signaling phenotype of fetal hepatocytes is altered when the cells are removed from theirin vivo milieu. In vivo, E19 hepatocytes showed a resistance to the S6K-activating effects of insulin and EGF and to the inhibitory effect of rapamycin on DNA synthesis. In contrast, primary cultures of E19 hepatocytes showed an intact response to hormonal activation of S6K1 and S6K2 as well as heightened sensitivity to the growth inhibitory effects of rapamycin. A similar in vivo toin vitro change in mitogenic signaling was seen in our studies on the developmental regulation of hepatic ERK1 and ERK2 (17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We have not established a mechanism for the uncoupling of proximal signaling from S6 kinase activation in developing liver. However, recent studies indicate a similar uncoupling of Akt activation and low levels of key proximal signaling molecules, including insulin receptor substrate-1 and Gab2. 2J. M. Boylan and P. A. Gruppuso, unpublished observations.As was the case for signaling to S6 kinases, fetal hepatocyte rapamycin resistance as measured by DNA synthesis was lost when hepatocytes were isolated and cultured. We observed an IC50 for both fetal and adult cells that was near 20 nm. The dose of rapamycin that we chose for our in vivo experiments was designed to achieve micromolar concentrations. Studies on “rapamycin-resistant” cancer cell lines have shown an IC50 in the micromolar range (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar).In summary, our results point to an alternative, S6 phosphorylation-independent pathway that supports fetal hepatocyte proliferation during late gestation. Our data indicate that phosphorylation of ribosomal protein S6 is not necessary for fetal hepatocyte proliferation in vivo. Given that translation of 5′-TOP mRNAs is thought to be essential for cellular proliferation, we conclude that fetal hepatocytes possess an alternative pathway for up-regulation of the translation of these genes. We found no evidence that this pathway is operative in adult hepatocytes. This conclusion is consistent with our previous studies (15Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar, 16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in that it suggests that mechanisms that mediate the proliferation of late gestation fetal hepatocytes are distinct from those that mediate the proliferation of mature hepatocytes in vivo. Finally, the rapamycin resistance displayed by a spectrum of cancer cells (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar) suggests that the mechanism for phospho-S6-independent hepatocyte proliferation in the late gestation fetal rat may prove relevant to liver carcinogenesis. For a cell to proliferate, it must up-regulate the biosynthetic apparatus needed to support cell growth. Studies have shown that the mRNA transcripts for all ribosomal proteins and protein synthesis elongation factors contain an unusual oligopyrimidine tract at their transcriptional start sites, commonly referred to as a 5′-TOP. The translation of these mRNAs has been shown to be dependent on S6K1 activation, presumably mediated by an increase in S6 phosphorylation (28Jefferies H.B. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (550) Google Scholar). Studies have demonstrated that 5′-TOP mRNA translation is selectively inhibited by the immunosuppressant rapamycin, a potent inhibitor of S6 kinase activity (29Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar). Our initial studies led to the unexpected finding that basal hepatic S6K1 activity in the late gestation fetal rat is lower than in adult animals. Given our prior observations demonstrating that late gestation fetal rat hepatocytes proliferate at a high rate both in vivo and in vitro (2Curran T.R.J. Bahner R.I.J. Oh W. Gruppuso P.A. Exp. Cell Res. 1993; 209: 53-57Crossref PubMed Scopus (49) Google Scholar, 3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar), the low basal S6K1 activity represents a dissociation between cell proliferation and activity of this key signaling enzyme (3Gruppuso P.A. Awad M. Bienieki T.C. Boylan J.M. Fernando S. Faris R.A. In Vitro Cell. Dev. Biol. 1997; 33: 562-568Crossref Scopus (27) Google Scholar). Furthermore, S6K1 activation in response to the in vivo administration of insulin or EGF was markedly attenuated in fetal liver compared with adult liver. This is reminiscent of our observations showing similar uncoupling of the ERK MAPK pathway in late gestation liver (16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We proceeded to demonstrate that, despite the low basal activity of S6K1, ribosomal protein S6 is indeed hyperphosphorylated in fetal liver. We considered S6K2 to be a candidate to account for the phosphorylation of S6 in fetal liver. Basal S6K2 activity was ∼2-fold higher in E19 liver than in adult liver, consistent with a role for S6K2 in maintaining S6 phosphorylation in vivo. However, the relatively low S6K2specific activity that we measured is entirely consistent with the possibility of an alternative mechanism for S6 hyperphosphorylation in fetal liver. One such explanation would be the existence of an alternative S6 kinase. Another possibility is a lower level of S6 phosphatase activity in fetal liver ribosomes. Studies by Olivieret al. (22Olivier A.R. Ballou L.M. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4720-4724Crossref PubMed Scopus (45) Google Scholar) indicate that the catalytic subunit of protein phosphatase-1 mediates the dephosphorylation of ribosomal protein S6. Low protein phosphatase-1 expression, decreased localization to ribosomes through decreased expression of associated subunits, or inhibition of ribosomal protein phosphatase-1 activity could serve to maintain S6 in a hyperphosphorylated state, even in the absence of excess kinase activity in fetal liver. To dissect the pathways accounting for the fetal liver S6 hyperphosphorylation, we administered rapamycin to intact animals. These studies were undertaken to test the hypothesis that fetal hepatocytes utilize an alternative, rapamycin-resistant pathway for S6 phosphorylation. Indeed, we found that rapamycin administration to E19 rats in situ did not inhibit DNA synthesis despite potent inhibition of S6K1 and S6K2. This led to analysis for the sensitivity of S6 phosphorylation to rapamycin and the unexpected finding that fetal hepatocyte proliferation in vivo could proceed following rapamycin administration even though S6 phosphorylation was markedly decreased. These results contrasted with those in adult animals. We employed a commonly used method for inducing adult hepatocytes to enter the cell cycle, a two-thirds hepatectomy (30Fausto N. Webber E.M. Arias A.M.B.J. Fausto N. Jacoby W.B. Schlacter D. Shafrits D.A. The Liver: Biology and Pathobiology. Raven Press, Ltd., New York1994: 1059-1084Google Scholar). This resulted in an increase in S6K1 activity as well as a marked increase in S6 phosphorylation. As anticipated, rapamycin effectively blocked in vivo S6 phosphorylation and hepatocyte DNA synthesis following partial hepatectomy. These results are consistent with the recently published report by Jiang et al. (25Jiang Y.-P. Ballou L.M. Lin R.Z. J. Biol. Chem. 2001; 276: 10943-10951Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) showing that rapamycin blocks activation of p70S6K in a dose-dependent manner and inhibits recovery of liver mass after a partial hepatectomy. There are precedents for the dissociation of S6 phosphorylation and cell proliferation. Kawasome et al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) performed studies that point to the existence of S6-independent mechanisms for 5′-TOP mRNA translation. Their S6K1 knockout murine embryonic stem cells did not show any S6 phosphorylation in vivo. However, 40% of total cellular eukaryotic elongation factor-1α mRNA, a 5′-TOP mRNA, was detected in polysomal fractions, indicating active eukaryotic elongation factor-1α translation. Prior to these studies, S6K1 activation was generally thought to be essential for G1/S transition. This was based on the work of Laneet al. (32Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar), who showed that microinjection of antibodies against S6K1 prevented the entry of cells into the cell cycle. Kawasomeet al. (31Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (159) Google Scholar) found that the S6K1 null embryonic stem cells proliferated well, albeit slower than parental cells, indicating that S6K1 is not essential for G1/S transition. Interestingly, rapamycin inhibited the proliferation of the S6K1 null cells to the same extent as the parental cells, indicating that events independent of S6K1 activation, but probably requiring mTOR activation, are critical for cell proliferation. This uncoupling of S6K1 activity and proliferation had previously been shown in activated T cells that were already cycling (33Terada N. Franklin R.A. Lucas J.J. Blenis J. Gelfand E.W. J. Biol. Chem. 1993; 268: 12062-12068Abstract Full Text PDF PubMed Google Scholar) as well as in erythroleukemic cell lines (34Calvo V. Wood M. Gjertson C. Vik T. Bierer B.E. Eur. J. Immunol. 1994; 24: 2664-2671Crossref PubMed Scopus (43) Google Scholar). Slavik et al. (35Slavik J.M. Lim D.-G. Burakoff S.J. Hafler D.A. J. Immunol. 2001; 166: 3201-3209Crossref PubMed Scopus (42) Google Scholar) recently found that p70S6K activation can be uncoupled from proliferation in both freshly isolated CD8+ T cells and CD8+ T cell clones, demonstrating for the first time rapamycin resistance in resting, nontransformed T cells. In studies by Withers et al. (36Withers D.J. Seufferlein T. Mann D. Garcia B. Jones N. Rozengurt E. J. Biol. Chem. 1997; 272: 2509-2514Crossref PubMed Scopus (42) Google Scholar) employing Swiss 3T3 cells, p70S6K activation was dissociated from DNA synthesis. Although rapamycin could inhibit both bombesin-mediated p70S6K activation and [3H]thymidine incorporation, the combination of bombesin plus insulin was able to stimulate DNA synthesis and cell cycle progression in the presence of rapamycin despite inhibition of p70S6K activity. These results were interpreted as indicating the presence of a rapamycin-insensitive mitogenic pathway in Swiss 3T3 cells. Another conclusion from our studies is that the signaling phenotype of fetal hepatocytes is altered when the cells are removed from theirin vivo milieu. In vivo, E19 hepatocytes showed a resistance to the S6K-activating effects of insulin and EGF and to the inhibitory effect of rapamycin on DNA synthesis. In contrast, primary cultures of E19 hepatocytes showed an intact response to hormonal activation of S6K1 and S6K2 as well as heightened sensitivity to the growth inhibitory effects of rapamycin. A similar in vivo toin vitro change in mitogenic signaling was seen in our studies on the developmental regulation of hepatic ERK1 and ERK2 (17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We have not established a mechanism for the uncoupling of proximal signaling from S6 kinase activation in developing liver. However, recent studies indicate a similar uncoupling of Akt activation and low levels of key proximal signaling molecules, including insulin receptor substrate-1 and Gab2. 2J. M. Boylan and P. A. Gruppuso, unpublished observations. As was the case for signaling to S6 kinases, fetal hepatocyte rapamycin resistance as measured by DNA synthesis was lost when hepatocytes were isolated and cultured. We observed an IC50 for both fetal and adult cells that was near 20 nm. The dose of rapamycin that we chose for our in vivo experiments was designed to achieve micromolar concentrations. Studies on “rapamycin-resistant” cancer cell lines have shown an IC50 in the micromolar range (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar). In summary, our results point to an alternative, S6 phosphorylation-independent pathway that supports fetal hepatocyte proliferation during late gestation. Our data indicate that phosphorylation of ribosomal protein S6 is not necessary for fetal hepatocyte proliferation in vivo. Given that translation of 5′-TOP mRNAs is thought to be essential for cellular proliferation, we conclude that fetal hepatocytes possess an alternative pathway for up-regulation of the translation of these genes. We found no evidence that this pathway is operative in adult hepatocytes. This conclusion is consistent with our previous studies (15Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar, 16Boylan J.M. Gruppuso P.A. Cell Growth Differ. 1996; 7: 1261-1269PubMed Google Scholar, 17Boylan J.M. Gruppuso P.A. J. Biol. Chem. 1998; 273: 3784-3790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in that it suggests that mechanisms that mediate the proliferation of late gestation fetal hepatocytes are distinct from those that mediate the proliferation of mature hepatocytes in vivo. Finally, the rapamycin resistance displayed by a spectrum of cancer cells (37Hosoi H. Dilling M.B. Liu L.N. Danks J.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar) suggests that the mechanism for phospho-S6-independent hepatocyte proliferation in the late gestation fetal rat may prove relevant to liver carcinogenesis. We greatly appreciate the assistance of Theresa Bienieki and Yang-Si Ou in the performance of these studies. We also thank Thomas Radimerski for helpful advice on the two-dimensional gel electrophoresis of ribosomal proteins." @default.
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W2080646585 title "Ribosomal Protein S6 Phosphorylation and Function during Late Gestation Liver Development in the Rat" @default.
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