Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.

Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was upregulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.     

Requirement for a hsp90 chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced cyclin D2 expression in mature B lymphocytes

A requirement for cyclin D2 in G(1)-to-S phase progression has been definitively established in mature B cells stimulated via the B cell antigen receptor (BCR). However, the identity of constituents of the BCR signaling cascade that leads to cyclin D2 accumulation remains incomplete. We report that inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked BCR-induced activation of extracellular signal-regulated kinase (ERK). Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate BCR-induced cyclin D2 expression at the mRNA and protein levels. Disruption of endogenous heat shock protein 90 (hsp90) function with geldanamycin abrogated BCR-induced cyclin D2 expression and proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of BCR signaling. These results suggest that the MEK1/2-ERK pathway is essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase- or protein kinase C-dependent pathways.     

The role of collagen structure in mitogen stimulation of ERK,cyclin D1 expression,and G1-S progression in rat hepatocytes

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.     

Differential expression of D-type G1 cyclins during mouse development and liver regeneration in vivo

D-type Gl cyclins are the primary cell cycle regulators of G1/S transition in eukaryotic cells, and are differentially expressed in a variety of cell lines in vitro. Little is known, however, about the expression patterns of D-type G1 cyclins in normal mouse in vivo. Thus, in the present study, tissue-specific expressions of cyclin D1 and D3 genes were examined in several tissues derived from adult male mice, and stage-specific expression of cyclin genes was studied in brain, liver, and kidney of developing mice from embryonic day 13 to postnatal day 11. Cell cycle-dependent expression of cyclins was also examined in regenerating livers following partial hepatectomy. Our results indicate that (l) cyclins Dl and D3 are expressed in a tissue-specific manner, with cyclin Dl being highly expressed in kidney and D3 in thymus; (2) cyclin D3 mRNA is abundantly expressed in young proliferating tissues and is gradually reduced during development, whereas cyclin Dl mRNA fluctuates during development; and (3) compensatory regeneration of liver induces cyclin Dl gene expression 12 hr after partial hepatectomy, and cyclin D3 gene expression from 36 to 42 hr (at the time of G1/S transition). In conclusion, this study indicates that cyclin D1 and D3 genes are differentially expressed in vivo in a tissue-specific, developmental stage-dependent, and cell cycle-dependent manner. © 1996 Wiley-Liss, Inc.     

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.     

MEK1 and MEK2, different regulators of the G1/S transition

The ERK cascade is activated by hormones, cytokines, and growth factors that result in either proliferation or growth arrest depending on the duration and intensity of the ERK activation. Here we provide evidence that the MEK1/ERK module preferentially provides proliferative signals, whereas the MEK2/ERK module induces growth arrest at the G1/S boundary. Depletion of either MEK subtype by RNA interference generated a unique phenotype. The MEK1 knock down led to p21cip1 induction and to the appearance of cells with a senescence-like phenotype. Permanent ablation of MEK1 resulted in reduced colony formation potential, indicating the importance of MEK1 for long term proliferation and survival. MEK2 deficiency, in contrast, was accompanied by a massive induction of cyclin D expression and, thus, CDK4/6 activation followed by nucleophosmin hyperphosphorylation and centrosome over-amplification. Our results suggest that the two MEK subtypes have distinct ways to contribute to a regulated ERK activity and cell cycle progression.     

Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Impaired hepatocyte DNA synthetic response posthepatectomy in insulin-like growth factor binding protein 1-deficient mice with defects in C/EBP beta and mitogen-activated protein kinase/extracellular signal-regulated kinase regulation

After a two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. One of the most rapidly and highly induced genes and proteins in regenerating liver is insulin-like growth factor binding protein 1 (IGFBP-1), a secreted protein that may modulate the activities of insulin-like growth factors (IGFs) or signal via IGF-independent mechanisms. To assess the functional role of IGFBP-1 in liver regeneration, mice with a targeted disruption of the IGFBP-1 gene were generated. Although IGFBP-1(-/-) mice demonstrated normal development, they had abnormal liver regeneration after partial hepatectomy, characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis. The abnormal regenerative response was associated with blunted activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and a reduced induction of C/EBP beta protein expression posthepatectomy. Like cell cycle abnormalities observed in hepatectomized C/EBP beta(-/-) mice, cyclin A and cyclin B1 expression was delayed and reduced in IGFBP-1(-/-) livers, whereas cyclin D1 expression was normal. Treatment of IGFBP-1(-/-) mice with a preoperative dose of IGFBP-1 induced MAPK/ERK activation and C/EBP beta expression, suggesting that IGFBP-1 may support liver regeneration at least in part via its effect on MAPK/ERK and C/EBP beta activities. These findings are the first demonstration of the involvement of IGFBP-1 in the regulation of in vivo mitogenic signaling pathways.     

ERK activation and mitogenesis in human airway smooth muscle cells

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.     

Inhibition of doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib--the role of extracellular signal-regulated kinase counteraction

A multikinase inhibitor of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.     

Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes.

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.     

Growth-related signaling regulates activation of telomerase in regenerating hepatocytes

Although there have been many reports on the relationship between activation of telomerase and carcinogenesis, the role of telomerase in normal cellular growth is still unclear. In this study, we analyzed the relationship between upregulation of telomerase activity and cell cycle progression during the liver regeneration process by using an in vivo mouse two-thirds partial hepatectomy (PH) model as well as by using in vitro hepatocyte culture systems. Furthermore, we also investigated the effects of growth factors on telomerase activity during liver regeneration and the influence of MAPK pathway inhibitors (MEK inhibitors PD98059 and U0126; p38 MAPK inhibitor SB203580) on the telomerase activity of regenerating hepatocytes in vitro. An upregulation of the telomerase activity was found at 24 h after PH, and thereafter an increase in the S-phase fraction was observed at 36-48 h. There was no remarkable change in the telomere length after PH. Preoperative treatment with EGF and HGF increased the in vivo telomerase activity. In a hepatocyte primary culture, the upregulation of the telomerase activity required the presence of EGF, and this upregulation was accelerated by the addition of HGF. A remarkable activation of p44/42 MAPK was seen but no such activation of p38 MAPK was observed at 48 h after PH. Although SB203580 had no effect on the telomerase activity of regenerating hepatocytes, treatment with MEK inhibitors (PD 98059, U0126) significantly repressed the telomerase activity. In conclusion, the telomerase activity is upregulated before hepatocytes enter the S phase, and both EGF and HGF play important roles in this step. In addition, the activation of the p44/42 MAPK pathway seems to play an essential role in telomerase upregulation during the liver regeneration process.     

Interruption of the MEK/ERK signaling cascade promotes dihydroartemisinin-induced apoptosis in vitro and in vivo

Artemisinin, the active principle of the Chinese medicinal herb Artemisia annua, and its derivatives (i.e. dihydroartemisinin, DHA) were reported to exhibit anti-tumor activity both in vitro and in vivo. The purpose of the present study was to investigate the functional role of Mitogen-Activated Protein Kinase (MEK)/Extracellular signal-regulated protein Kinase (ERK) signaling cascade in dihydroartemisinin (DHA)-induced apoptosis in human leukemia cells in vitro and anti-leukemic activity in vivo. Human leukemia cells were treated with DHA in dose- and time-dependent manners, after which apoptosis, caspase activation, Mcl-1 expression, and cell signaling pathways were evaluated. Parallel studies were performed in AML and ALL primary human leukemia cells. In vivo anti-leukemic activity mediated by DHA was also investigated using U937 xenograft mouse model. Exposure of DHA resulted in a pronounced increase in apoptosis in both transformed and primary human leukemia cells but not in normal peripheral blood mononuclear cells. DHA-induced apoptosis was accompanied by caspase activation, cytochrome c release, Mcl-1 down-regulation, as well as MEK/ERK inactivation. Pretreatment with MEK inhibitor PD98059, which potentiated DHA-mediated MEK and ERK inactivation, intensified DHA-mediated apoptosis. Conversely, enforced expression of a constitutively active MEK1 attenuated DHA-induced apoptosis. Furthermore, DHA-mediated inhibition of tumor growth of mouse U937 xenograft was associated with induction of apoptosis and inactivation of ERK. The findings in the present study showed that DHA-induced apoptosis in human leukemia cells in vitro and exhibited an anti-leukemic activity in vivo through a process that involves MEK/ERK inactivation, Mcl-1 down-regulation, culminating in cytochrome c release and caspase activation.     

Raf-induced cell cycle progression in human TF-1 hematopoietic cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to beta-estradiol-regulated DeltaRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21(Cip1), which are associated with G(1) progression. Activated DeltaRaf-1:ER and DeltaA-Raf:ER but not DeltaB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16(Ink4a), a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16(Ink4a) suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.     

Involvement of IQGAP3, a regulator of Ras/ERK‐related cascade,in hepatocyte proliferation in mouse liver regeneration and development

The spatio‐temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42‐binding proliferation factor associated with cell–cell contacts in epithelial‐type cells. In parallel, the expression/localization of Rac/Cdc42‐binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki‐67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell–cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl₄‐induced (but not in partial hepatectomy‐induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell–cell contact‐related proliferating signaling events. On the other hand, the Rac/Cdc42‐binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl₄ injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development. J. Cell. Physiol. 220: 621–631, 2009. © 2009 Wiley‐Liss, Inc.