The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial 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.     

ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells

ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.     

E2F-1 has properties of a radiosensitizer and its regulation by cyclin A kinase is required for cell survival of fibrosarcoma cells lacking p53.

Negative regulation of E2F-1 DNA binding function by cyclin A kinase represents part of an S-phase checkpoint control system that, when activated, leads to apoptosis. In this study, we examined the cellular sensitivity and resistance of isogenic mouse fibrosarcoma cell lines, differing primarily in their p53 status, to ectopic expression of wild-type (wt) E2F-1 and cyclin A kinase binding-defective mutants of it. We found that E2F-1 (wt) potently affected the survival of p53+/+ tumor cells but not that of p53-/- cells. In contrast, expression of cyclin A kinase binding-defective E2F-1 species interfered with cell survival of fibrosarcoma cells irrespective of their p53 status. Finally, expression of E2F-1 (wt) in p53-/- fibrosarcoma cells enhanced the cytotoxic effect of ionizing radiation in vitro and in vivo in a mouse tumor model. These results suggest that E2F-1-dependent activation of an S-phase checkpoint is p53 independent and that E2F-1 possesses radiosensitizing properties in the absence of p53.     

Inhibition of benzo(a)pyrene-induced cell cycle progression by all-trans retinoic acid partly through cyclin D1/E2F-1 pathway in human embryo lung fibroblasts

Benzo(a)pyrene [B(a)P] is a potent environmental carcinogen, which induces cell cycle changes. All-trans retinoic acid (ATRA) is a promising agent in prevention and treatment of human cancers. In the present study, we investigated the inhibition of B(a)P-induced cell cycle progression by ATRA in human embryo lung fibroblast (HELF). Our results showed that after treatment with B(a)P, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF. There were almost no changes of CDK4 and E2F-4 expression by treatment with B(a)P. As expected, pretreatment with ATRA could efficiently decrease B(a)P-induced overexpression of cyclin D1 and E2F-1. In a further study, we stably transfected antisense cyclin D1 and antisense CDK4 plasmid into HELF. The inhibition of cyclin D1 expression and the inhibition of CDK4 expression significantly impaired the B(a)P-induced overexpression of E2F-1 respectively. Pretreatment with ATRA, cells expressing antisense cyclinD1 or antisense CDK4 showed a lesser decrease of B(a)P-induced overexpression of E2F-1 compared with similarly treated HELF. Furthermore, flow cytometry analysis showed that B(a)P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B(a)P-induced cell cycle progression by an accumulation of cells in the G(1) phase. It was suggested that ATRA could block B(a)P-induced cell cycle promotion partly through the cyclin D1/E2F-1 pathway in HELF.     

Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage

Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.     

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.     

Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.     

Cyclin D1 determines mitochondrial function in vivo

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.     

Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factor beta1

To determine if Neu is dominant over transforming growth factor beta (TGF-beta), we crossed mouse mammary tumor virus (MMTV)-Neu mice with MMTV-TGF-beta1(S223/225) mice expressing active TGF-beta1 in the mammary gland. Bigenic (NT) and Neu-induced mammary tumors developed with a similar latency. The bigenic tumors and their metastases were less proliferative than those occurring in MMTV-Neu mice. However, NT tumors exhibited less apoptosis and were more locally invasive and of higher histological grade. NT mice exhibited more circulating tumor cells and lung metastases than Neu mice, while NT tumors contained higher levels of phosphorylated (active) Smad2, Akt, mitogen-activated protein kinase (MAPK), and p38, as well as vimentin content and Rac1 activity in situ than tumors expressing Neu alone. Ex vivo, NT cells exhibited higher levels of P-Akt and P-MAPK than Neu cells. These were inhibited by the TGF-beta inhibitor-soluble TGF-beta type II receptor (TbetaRII:Fc), suggesting they were activated by autocrine TGF-beta. TGF-beta stimulated migration of Neu cells into surrounding matrix, while the soluble TGF-beta inhibitor abrogated motility and invasiveness of NT cells. These data suggest that (i) the antimitogenic and prometastatic effects of TGF-beta can exist simultaneously and (ii) Neu does not abrogate TGF-beta-mediated antiproliferative action but can synergize with TGF-beta in accelerating metastatic tumor progression.