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.