Growth factors elicit their biological effects by activating a complex network of receptors and signaling pathways. Activation of transmembrane tyrosine kinases by serum or polypeptide growth factors results in the transit of cells through the G 1 phase of the cell cycle into S-phase. Several lines of evidence suggest that the D-type cyclins and their associated kinases (Cdks) 1 are among the targets of these growth signals (1). The D-type cyclins, D1, D2 and D3, are closely related proteins whose expression is induced by mitogens and growth factors (2-6) and down-regulated by growth factor deprivation or by antimitogens (7,8). The D-type cyclins associate with cyclindependent protein kinase Cdk4 or Cdk6 to form an active complex that phosphorylates and inactivates the retinoblastoma protein, pRb (9, 10). Inhibition of cyclin D1 expression either by antisense methodology or antibody microinjection lengthens the duration of the G 1 phase and causes a reduction in proliferation (11,12). Aberrant overexpression of D-type cyclins resulting from upstream growth factor receptor activation, gene amplification or rearrangement, or an increase in mRNA stability seems to be a common feature of a number of human cancers and may reduce the cell's dependence on physiologic growth stimuli (13-16).Changes in cyclin D expression integrate the proliferative effects of an array of extracellular factors, including cytokines, polypeptide growth factors, and steroid hormones (2-4, 7). Cellular stress results in the loss of cyclin D1 expression, with a concomitant arrest in the G 1 phase of the cell cycle (8, 17). The networks of pathways responsible for the transduction of these signals are complex and not completely understood. There is some evidence suggesting that a Ras-and MAP kinasedependent signaling pathway is involved. Expression of activated Ras is associated with the increased expression of cyclin D1 in both epithelial cells (12) and fibroblasts (11). Moreover, in the absence of growth factors, activation of the Raf1 3 MEK 3 MAP kinase pathway has been shown to be sufficient to induce cyclin D1 transcription (5). Herbimycin A is a natural product that binds to a specific site in Hsp90 and causes the degradation of transmembrane tyrosine protein kinases, Raf1, and steroid hormone receptors (18 -23). We found that treatment of tumor cells with this drug causes a decrease in the expression of D-type cyclins and an Rb-dependent G 1 block. 2 We report here that the reduction in the level of D-type cyclins induced by herbimycin A is due to inhibition of translation of cyclin D mRNAs. Furthermore, the increase in the level of D-type cyclins in cells treated with serum is due to an increase in the translation of their mRNAs. These effects are due to the regulation of a PI 3-kinase/Akt kinase-dependent, Raf1-and MAP kinase-independent pathway. This pathway is activated by serum and is blocked by the drug herbimycin A. EXPERIMENTAL PROCEDURESCells and Antibodies-Colo205, a human colon carcinoma cell line, and MCF7, a breast cancer c...

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The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear-encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear-encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O(2) consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC-1alpha, and a reduction in known PGC-1alpha target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor-induced increases in cellular energy demands.

Acutely transforming retrovirus AKT8 in rodent T-cell lymphoma (Akt) is a serine/threonine kinase that plays important roles in survival, cell-cycle progression, and cell proliferation, and has recently been implicated in collagen regulation. The aim of this study was to determine the role of Akt in collagen deposition by normal dermal fibroblasts, and to determine the sensitivity of cultured systemic sclerosis (SSc) fibroblasts to Akt inhibition. We show that blockade of Akt using pharmacological inhibitors, small interfering RNA (siRNA), and a dominant-negative Akt mutant led to inhibition of the basal type I collagen production. Furthermore, inhibition of Akt upregulated basal matrix metalloproteinase 1 (MMP1) production and reversed the inhibitory effect of transforming growth factor-beta (TGF-beta) on MMP1 gene expression. In addition, SSc fibroblasts were more sensitive to Akt inhibition, with respect to collagen and MMP1 production. These findings suggest that in human dermal fibroblasts, Akt has dual profibrotic effects, increasing collagen synthesis and decreasing its degradation via downregulation of MMP1. Akt could directly contribute to elevated collagen in SSc fibroblasts and it may represent an attractive target for therapy of SSc fibrosis.

CC, Wright GL. O2-sensing signal cascade: clamping of O2 respiration, reduced ATP utilization, and inducible fumarate respiration. Am J Physiol Cell Physiol 295: C29 -C37, 2008. First published May 7, 2008 doi:10.1152/ajpcell.00466.2007.-These studies explore the consequences of activating the prolyl hydroxylase (PHD) O2-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broadspectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O 2 consumption and a 300% increase in lactic acid production under basal conditions. This indicates a ϳ75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O2 consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O 2 consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHItreated cardiomyocytes. Silencing of hypoxia-inducible factor-1␣ (HIF-1␣) expression restored the ability of uncoupled PHI-treated myocytes to increase O 2 consumption; however, basal O2 uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O 2-sensing pathway is sufficient to actively "clamp" O 2 consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration. mitochondrial membrane potential; hibernation; cardioprotection ALTHOUGH OXYGENATION of lower eukaryotes is accomplished by simple diffusion, an extensive cardiovascular and respiratory system has evolved in higher animals to enable tissue and organ oxygenation. In turn, cells, and the tissue they comprise, closely monitor O 2 levels and initiate a complex response to restore homeostasis when lower O 2 concentrations are detected. These responses include release of vasodilator agents, expression of angiogenic factors, and upregulation of anaerobic glycolytic metabolism, among others. Many of the cellular responses to lower O 2 occur at concentrations higher than those limiting aerobic mitochondrial ATP production (38), an observation that first suggested the presence of a cellular O 2 -sensing mechanism distinct from the electron transport chain (ETC) or ATP depletion. More recently, a cellular O ...

The ETS1 transcription factor is a member of the Ets family of conserved sequence-specific DNA-binding proteins. ETS1 has been shown to play important roles in various cellular processes such as proliferation, differentiation, lymphoid development, motility, invasion and angiogenesis. These diverse roles of ETS1 are likely to be dependent on specific protein interactions. To identify proteins that interact with ETS1, a yeast two-hybrid screen was conducted. Here, we describe the functional interaction between SP100 and ETS1. SP100 protein interacts with ETS1 both in vitro and in vivo. SP100 is localized to nuclear bodies and ETS1 expression alters the nuclear body morphology in living cells. SP100 negatively modulates ETS1 transcriptional activation of the MMP1 and uPA promoters in a dose-dependent manner, decreases the expression of these endogenous genes, and reduces ETS1 DNA binding. Expression of SP100 inhibits the invasion of breast cancer cells and is induced by Interferon-a, which has been shown to inhibit the invasion of cancer cells. These data demonstrate that SP100 modulates ETS1-dependent biological processes.

Background Eukaryotic Initiation Factor 4E-Binding Protein ( EIF4EBP1 , 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. Methods Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. Results Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. Conclusions These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation. Electronic supplementary material The online version of this article (10.1186/s12885-019-5667-4) contains supplementary material, which is available to authorized users.

Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.