Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3 beta phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3 beta phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3beta antibody. Endogenous IGF-I stimulated GSK-3 beta phosphorylation and inhibited GSK-3 beta activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3 beta phosphorylation and the resulting GSK-3 beta inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced beta-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3 beta inhibitor. Endogenous IGF-I inhibited beta-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3 beta activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.     

MICAL1 facilitates breast cancer cell proliferation via ROS‐sensitive ERK/cyclin D pathway

Molecule interacting with CasL 1 (MICAL1) is a multidomain flavoprotein mono‐oxygenase that strongly involves in cytoskeleton dynamics and cell oxidoreduction metabolism. Recently, results from our laboratory have shown that MICAL1 modulates reactive oxygen species (ROS) production, and the latter then activates phosphatidyl inositol 3‐kinase (PI3K)/protein kinase B (Akt) signalling pathway which regulates breast cancer cell invasion. Herein, we performed this study to assess the involvement of MICAL1 in breast cancer cell proliferation and to explore the potential molecular mechanism. We noticed that depletion of MICAL1 markedly reduced cell proliferation in breast cancer cell line MCF‐7 and T47D. This effect of MICAL1 on proliferation was independent of wnt/β‐catenin and NF‐κB pathways. Interestingly, depletion of MICAL1 significantly inhibited ROS production, decreased p‐ERK expression and unfavourable for proliferative phenotype of breast cancer cells. Likewise, MICAL1 overexpression increased p‐ERK level as well as p‐ERK nucleus translocation. Moreover, we investigated the effect of MICAL1 on cell cycle‐related proteins. MICAL1 positively regulated CDK4 and cyclin D expression, but not CDK2, CDK6, cyclin A and cyclin E. In addition, more expression of CDK4 and cyclin D by MICAL1 overexpression was blocked by PI3K/Akt inhibitor LY294002. LY294002 treatment also attenuated the increase in the p‐ERK level in MICAL1‐overexpressed breast cancer cells. Together, our results suggest that MICAL1 exhibits its effect on proliferation via maintaining cyclin D expression through ROS‐sensitive PI3K/Akt/ERK signalling in breast cancer cells.     

Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.     

Proliferation of intimal smooth muscle cells. Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with increased expression of cell cycle inhibitors

Basic fibroblast growth factor (FGF2) is a potent mitogen for medial smooth muscle cells and is necessary for their proliferation after balloon catheter injury; however, intimal smooth muscle cells do not require FGF2 for their proliferation, and they respond only weakly to exogenous FGF2. The present study examined the activation of extracellular signal-regulated kinase (ERK) signaling as well as the expression and activity of cell cycle proteins in FGF2-stimulated intimal smooth muscle cells. FGF2 activates ERKs 1 and 2, and Western blot analysis showed that cyclin D, cyclin E, and cyclin-dependent kinase (CDKs) 2 and 4 were expressed in intimal smooth muscle cells after FGF2 infusion. FGF2 stimulation, however, did not lead to phosphorylation of the retinoblastoma protein (Rb), CDK 2 activation, or expression of cyclin A. Western blot analysis showed that intimal smooth muscle cells express elevated levels of the cell cycle inhibitors p15(INK4b) and p27(Kip1), compared with medial smooth muscle cells, and that FGF2 stimulation does not reduce the level of these inhibitors. These studies suggest that despite activation of ERKs 1 and 2 and expression of the cell cycle activators, cyclin D and cyclin E, high levels of cell cycle inhibitors may inhibit cell cycle transit in FGF2-stimulated intimal smooth muscle cells.     

Roles of the ERK, JNK/AP-1/cyclin D1-CDK4 pathway in silica-induced cell cycle changes in human embryo lung fibroblast cells

Silica is a potent occupational fibrogenic agent capable of inducing lung fibrosis and many other lung diseases. Our current study focused on the signalling pathways regulating cell cycle changes in HELF (human embryo lung fibroblast) after silica (α-quartz) exposure. Our results showed silica exposure could lead to cell cycle changes. The cell cycle alternations were accompanied with overexpression of cyclin D1 and CDK4 (cyclin-dependent kinase 4) in a time-dependent manner. Silica exposure also decreased E2F-4 expression in HELF. These changes were blocked by overexpression of dominant-negative mutants of ERK (extracellular signal-regulated protein kinase) or the JNK (stress-activated c-Jun NH2-terminal kinase), respectively. Moreover, pretreatment of cells with curcumin, an activation of AP-1 (activator protein-1) inhibitor, inhibited silica-induced cell cycle alteration, the decreased expression of E2F-4 and overexpression of cyclin D1 and CDK4. Furthermore, both antisense cyclin D1 and antisense CDK4 can block silica-induced cell cycle changes. These results suggest that silica exposure can induce cell cycle changes, which may be mediated through ERK, JNK/AP-1/cyclin D1-CDK4-dependent pathway.     

Phosphatidylinositol 3-kinase activity regulates alpha -thrombin-stimulated G1 progression by its effect on cyclin D1 expression and cyclin-dependent kinase 4 activity

In this study, we present evidence that PI 3-kinase is required for alpha-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G(1) phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the alpha-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G(1).     

Activation of Transient Receptor Potential Vanilloid 4 Promotes the Proliferation of Stem Cells in the Adult Hippocampal Dentate Gyrus

Neurogenesis plays an important role in adult hippocampal function, and this process can be modulated by intracellular calcium. The activation of transient receptor potential vanilloid 4 (TRPV4) induces an increase in intracellular calcium concentration, but whether neurogenesis can be modulated by TRPV4 activation remains unclear. Here, we report that intracerebroventricular injection of the TRPV4 agonist GSK1016790A for 5 days enhanced the proliferation of stem cells in the hippocampal dentate gyrus (DG) of adult mice without affecting neurite growth, differentiation, or survival of newborn cells. GSK1016790A induced increases in the hippocampal protein levels of cyclin-dependent kinase (CDK) 6, CDK2, cyclin E1, and cyclin A2 but did not affect CDK4 and cyclin D1 expression. The phosphorylation of retinoblastoma protein (Rb) in hippocampi was enhanced in GSK1016790A-injected mice compared with control mice. Moreover, hippocampal protein levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were enhanced by GSK1016790A. Finally, GSK1016790A-enhanced proliferation was markedly blocked by a MAPK/ERK kinase or p38 MAPK antagonist (U0126 or SB203580, respectively). The increased protein levels of CDK2 and CDK6, as well as those of cyclin E1 and cyclin A2, in GSK1016790A-injected mice were substantially reduced by co-injection of U0126 or SB203580. We conclude that TRPV4 activation results in the proliferation of stem cells in the adult hippocampal DG, which is likely mediated through ERK1/2 and p38 MAPK signaling to increase the expression of CDKs (CDK6 and CDK2) and cyclins (cyclin E1 and A2), phosphorylate Rb consequently, and accelerate the cell cycle ultimately.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-( >3 hr) and dose-(> 25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1-S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10(-6) M), Akt (Akt inhibitor, 10(-5) M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10(-5) M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways.     

G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells

Ovarian cancer is one of the most common cancers among women. Recent studies demonstrated that the gene encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is frequently amplified in ovarian cancer cells. PI3K is involved in multiple cellular functions, including proliferation, differentiation, antiapoptosis, tumorigenesis, and angiogenesis. In this study, we demonstrate that the inhibition of PI3K activity by LY-294002 inhibited ovarian cancer cell proliferation and induced G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 4, CDC25A, and retinoblastoma phosphorylation at Ser(780), Ser(795), and Ser(807/811). Expression of CDK6 and beta-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16(INK4a) was induced by the PI3K inhibitor, whereas steady-state levels of p21(CIP1/WAF1) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G(1) cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G(1) cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G(1) progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells.     

Invited review: the circle of life: cell cycle regulation in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G(1) progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.     

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

Anti-proliferative effects of γ-tocotrienol on mammary tumour cells are associated with suppression of cell cycle progression

Objectives:  Previous studies have shown that γ-tocotrienol induces potent anti-proliferative effects on +SA mammary tumour cells in culture; here, investigations have been conducted to determine its effects on intracellular signalling proteins involved in regulating cell cycle progression.
Materials and methods:  +SA cells were maintained in mitogen-free defined media containing 0 or 4 μ m γ-tocotrienol, for 48 h to synchronize cell cycle in G₀ phase, and then they were exposed to 100 ng/ml EGF to initiate cell cycle progression. Whole cell lysates were collected at various time points from each treatment group and were prepared for Western blot analysis.
Results and conclusions:  Treatment with 4 μ m γ-tocotrienol significantly inhibited +SA cell proliferation over a 4-day culture period. Moreover, this treatment resulted in a relatively large reduction in cyclin D1, cyclin dependent kinase (CDK)4, CDK2 and CDK6 levels, between 4 and 24 h after EGF exposure. Tocotrienol treatment also resulted in a relatively large increase in CDK inhibitor (CKI) p27, prior to and after EGF exposure, but had little effect on levels of CKIs, p21 and p15. Tocotrienol treatment also induced a large relative reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. These findings strongly suggest that anti-proliferative effects of γ-tocotrienol are associated with reduction in cell cycle progression from G₁ to S, as evidenced by increased p27 levels, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6 and phosphorylated Rb levels.     

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.     

The N-terminal 24 amino acids of the p55 gamma regulatory subunit of phosphoinositide 3-kinase binds Rb and induces cell cycle arrest

Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55alpha and p55gamma) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55gamma in the nucleus. The 24-amino-acid N-terminal end of p55gamma, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55gamma inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G0/G1 phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55gamma on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55gamma with Rb and show that modification of this association can lead to cell cycle arrest.     

Oncostatin M induces growth arrest of skeletal muscle cells in G1 phase by regulating cyclin D1 protein level

Oncostatin M (OSM), an IL-6 family cytokine, either inhibits or enhances the growth of cells depending on cell type. Here, we report that OSM inhibits proliferation of skeletal muscle cells by blocking cell cycle progression from G(1) to S phase. OSM treatment significantly reduced levels of cyclin D1 protein and phosphorylation of retinoblastoma protein (Rb) at Ser-795, a CDK4-specific phosphorylation site. The OSM-induced cyclin D1 reduction correlated with decreased amount of the cyclin D1/p27 Kip1 complex and increased amounts of the CDK2/p27 Kip1 complex, resulting in inhibition of CDK2 activity. Results obtained with lactacystin, a proteasome inhibitor, demonstrated that cyclin D1 reduction occurred through ubiquitin/proteasome proteolysis. In addition, activation of STAT3, but not STAT1, is likely to regulate OSM-induced cyclin D1 reduction. Dominant negative (DN)-STAT3 blocked OSM-induced cyclin D1 reduction, and constitutively active-STAT3 also induced cyclin D1 reduction. These results suggest that OSM arrests skeletal muscle cell growth at the G1/S checkpoint and that this response occurs by an ubiquitin/proteasome-dependent cyclin D1 protein reduction which is regulated by STAT3.     

Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells

Stimulation of the breast cancer-derived MCF-7S cell line with insulin-like growth factor I (IGF-I; 20 ng/ml) leads to enhanced expression of cyclin D1, hyperphosphorylation of pRb, DNA synthesis, and cell division. 17beta-Estradiol (E(2); 10(-9) m) is not able to stimulate proliferation of MCF-7S cells, although addition of E(2) to serum-starved cells does result in induction of cyclin D1. However, in combination with submitogenic amounts of IGF-I (2 ng/ml), E(2) induces cell proliferation. We have previously shown that the synergistic action of E(2) and IGF-I emanates from the ability of both hormones to induce cyclin D1 expression and that IGF-I action is required to induce activity of the cyclin D1-CDK4 complex, which triggers cell cycle progression. Here, we show that IGF-I (but not E(2)) is able to induce nuclear accumulation of cyclin D1 by a phosphatidylinositol 3-kinase-dependent mechanism. Nuclear accumulation of cyclin D1 and cell cycle progression were also observed when LiCl, a known inhibitor of GSK3beta, was added to E(2)-stimulated cells. Thus, inhibition of GSK3beta activity appears to trigger nuclear accumulation of cyclin D1 and cell cycle progression. This notion was confirmed by overexpression of constitutively active GSK3beta, which blocks IGF-I-induced nuclear accumulation of cyclin D1 as well as S phase transition.