Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase Balpha

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKBα in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKBα and Akt2/PKBβ by ectopic expression of Akt1/PKBα but not Akt2/PKBβ. Akt1/PKBα was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKBα-deficient cells, but was restored after forced expression of Akt1/PKBα. Moreover, expression of p27^Kip1, an inhibitor of the cell cycle, was down regulated in an Akt1/PKBα-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKBα isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27^Kip1.     

Non-CDK-bound p27 (p27) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways

p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27^NCDK) but the nature of p27^NCDK has remained unresolved. Here we demonstrate that the epitope recognized by the p27^NCDK-specific antibody resides in the p27 CDK-interaction domain and that p27^NCDK is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27^NCDK levels whereas total p27 levels are unaffected. p27^NCDK, but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27^NCDK responses to metabolic stress and PI3K inhibition. These results indicate that p27^NCDK is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.     

PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27Kip1

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We previously demonstrated that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in primary MKs in a PI3K dependent fashion. UT-7/TPO, a megakaryocytic cell line, stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to reduce p27Kip1 expression after TPO stimulation, and fail to induce p27Kip1 expression following TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In addition, the number of MKs is significantly increased in bone marrow from Foxo3a-deficient mice. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings indicate that the PI3K/Akt/FOXO3a/p27Kip1 pathway contributes to normal TPO-induced MK proliferation.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.     

Pristimerin-induced uveal melanoma cell death via inhibiting PI3K/Akt/FoxO3a signalling pathway

Uveal melanoma (UM) is a highly invasive intraocular malignancy with high mortality. Presently, there is no FDA-approved standard for the treatment of metastatic UM. Pristimerin is a natural quinine methide triterpenoid compound with anti-angiogenic, anti-cancer and anti-inflammatory activities. However, Pristimerin potential cytotoxic effect on UM was poorly investigated. In the present study, we found the migration and invasion of UM-1 cells were inhibited by Pristimerin which also caused a rapid increase of ROS, decreased mitochondrial membrane potential, induced the accumulation of cells in G0/G1 phase, ending with apoptotic cell death. Pristimerin inhibited Akt and FoxO3a phosphorylation and induced nuclear accumulation of FoxO3a in UM-1 cells, increased the expression of pro-apoptotic proteins Bim、p27^Kip1, cleaved caspase-3, PARP and Bax, and decreased the expression of Cyclin D1 and Bcl-2. LY294002 or Akt-siRNA inhibited the PI3K/Akt/FoxO3a pathway and promoted the Pristimerin-induced apoptosis, while Pristimerin effects were partially abolished in FoxO3a knockdown UM-1 cell cultures. Taken together, present results showed that Pristimerin induced apoptotic cell death through inhibition of PI3K/Akt/FoxO3a pathway in UM-1 cells. These findings indicate that Pristimerin may be considered as a potential chemotherapeutic agent for patients with UM.     

Deficiency of cyclin-dependent kinase inhibitors p21 and p27 accelerates atherogenesis in apolipoprotein E-deficient mice

Cyclin-dependent kinase inhibitors, p21^Cip1 and p27^Kip1, are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21^Cip1 or p27^Kip1 in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE^−/− aortae, both apoE^−/−/p21^−/− and apoE^−/−/p27^−/− aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27^Kip1 accelerated plaque formation significantly more than p21^−/− in apoE^−/− mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21^Cip1 and p27^Kip1 accelerates atherogenesis in apoE^−/− mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.     

Streptogramin- and tetracycline-responsive dual regulated expression of p27(Kip1) sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27^Kip1 in sense (p27^Kip1S) and antisense (p27^Kip1AS) orientation. Exclusive expression of p27^Kip1S resulted in complete G₁-phase-specific growth arrest, while expression of only p27^Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27^Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27^Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27^Kip1 failed to provide the expected G₁-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.     

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.     

High glucose regulates cyclin D1/E of human mesenchymal stem cells through TGF‐β1 expression via Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signal pathways

The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill‐defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D ‐glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [³H]‐thymidine incorporation and cell‐cycle regulatory protein expression levels compared with 5 mM D ‐glucose or 25 mM L ‐glucose. In addition, high glucose increased transforming growth factor‐β1 (TGF‐β₁) mRNA and protein expression levels. High glucose‐induced cell‐cycle regulatory protein expression levels and [³H]‐thymidine incorporation, which were inhibited by TGF‐β₁ siRNA transfection and TGF‐β₁ neutralizing antibody treatment. High glucose‐induced phosphorylation of protein kinase C (PKC), p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time‐dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10^?6 M; bisindolylmaleimide I, 10^?6 M), LY 294002 (PI3 kinase inhibitor, 10^?6 M), Akt inhibitor (10^?5 M), PD 98059 (p44/42 MAPKs inhibitor, 10^?5 M), SB 203580 (p38 MAPK inhibitor, 10^?6 M), and rapamycin (mTOR inhibitor, 10^?8 M) blocked the high glucose‐induced cellular proliferation and TGF‐β₁ protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF‐β₁ expression via Ca²⁺/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010 © 2010 Wiley‐Liss, Inc.     

Long‐term repopulation of aged bone marrow stem cells using young Sca‐1 cells promotes aged heart rejuvenation

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca‐1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca‐1⁺ or Sca‐1^? cells from young (2–3 months) or aged (18–19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca‐1⁺, young Sca‐1^?, old Sca‐1⁺, and old Sca‐1^?. Four months later, expression of rejuvenation‐related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase‐related protein (DNA‐PKcs, TRF‐2) expression, whereas expression of senescence‐related genes (p16^INK4a, P19^ARF, p27^Kip1) and proteins (p16^INK4a, p27^Kip1) was decreased in Sca‐1⁺ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP^?CD31⁺) but not cardiomyocytes were the primary cell type rejuvenated by young Sca‐1⁺ cells as shown by improved proliferation, migration, and tubular formation abilities. C‐X‐C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP⁺) cells isolated from young Sca‐1⁺ chimeric hearts. Protein expression of Cxcr4, phospho‐Akt, and phospho‐FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca‐1⁺ group. Reconstitution of aged BM with young Sca‐1⁺ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.     

RAD 001 (everolimus) prevents mTOR and Akt late re‐activation in response to imatinib in chronic myeloid leukemia

The mammalian target of rapamycin (mTOR) is one target of BCR‐ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug‐resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR‐phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re‐activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de‐phosphorylation of Akt at Ser⁴⁷³ in the hydrophobic motif of C‐terminal tail required for Akt full activation and precludes Akt re‐phosphorylation in response to IM. Moreover, RAD 001‐induced inhibition of Akt causes the de‐phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14‐3‐3 binding sites, TSC2 release from 14‐3‐3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin‐dependent kinase [CDK] inhibitor p27^Kip1, the stabilization of p27^Kip1 ligand with CDK2, and the G₀/G₁ arrest). RAD 001 cytotoxicity on cells not expressing the BCR‐ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect. J. Cell. Biochem. 109: 320–328, 2010. © 2009 Wiley‐Liss, Inc.     

CXCR4‐independent rescue of the myeloproliferative defect of the gata1low myelofibrosis mouse model by Aplidin®

The discovery of JAK2 mutations in Philadelphia‐negative myeloproliferative neoplasms has prompted investigators to evaluate mutation‐targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1^low mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27^Kip1 activity and is treatable by Aplidin®, a cyclic depsipeptide that activates p27^Kip1 in several cancer cells. Aplidin® restored expression of Gata1 and p27^Kip1 in Gata1^low hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF‐β/VEGF levels released in the microenvironment by immature Gata1^low megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin®‐treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1^low progenitor cells remained low. These results indicate that Aplidin® effectively alters the natural history of myelofibrosis in Gata1^low mice and suggest this drug as candidate for clinical evaluation in PMF. J. Cell. Physiol. 225: 490–499, 2010. © 2010 Wiley‐Liss, Inc.     

Attenuation of cell cycle regulator p27<Superscript>Kip1</Superscript> expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro

Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27^Kip1 has been implicated in this context in epithelial cells. We cloned and sequenced p27^Kip1 of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27^Kip1 downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27^Kip1 is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.