HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

Download Full-text

β1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal α-actin promoter in myoblasts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.6.h1736 ◽

2000 ◽

Vol 278 (6) ◽

pp. H1736-H1743 ◽

Cited By ~ 23

Author(s):

Lei Wei ◽

Wei Zhou ◽

Lu Wang ◽

Robert J. Schwartz

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Serum Response Factor ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Muscle Gene Expression ◽

Actin Promoter ◽

Muscle Gene ◽

Negative Mutant

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c- fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal α-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal α-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the β1-integrin stimulated RhoA-dependent activation of the α-actin promoter. The muscle-specific isoform β1Dselectively activated the α-actin promoter in concert with RhoA but inhibited the c- fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the α-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited α-actin promoter activity induced by RhoA. These results suggest that RhoA, β1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

Download Full-text

Regulation of Id Gene Expression by Type I Insulin-Like Growth Factor: Roles of STAT3 and the Tyrosine 950 Residue of the Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.21.16.5447-5458.2001 ◽

2001 ◽

Vol 21 (16) ◽

pp. 5447-5458 ◽

Cited By ~ 18

Author(s):

Marco Prisco ◽

Francesca Peruzzi ◽

Barbara Belletti ◽

Renato Baserga

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Growth Factor Receptor ◽

Cell Types ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Type I ◽

Insulin Like Growth Factor ◽

Proliferation And Differentiation ◽

Igf I

ABSTRACT Id proteins are known to play important roles in the proliferation and differentiation of many cell types. The type 1 insulin-like growth factor receptor (IGF-IR), activated by its ligand, induces the differentiation of 32D IGF-IR cells, a murine hematopoietic cell line, expressing a human IGF-IR. Expression in 32D IGF-IR cells of a dominant negative mutant of Stat3 (DNStat3) inhibits IGF-I-mediated differentiation. DNStat3 causes a dramatic increase in Id2 gene expression. This increase, however, is IGF-I dependent and is abrogated by a mutation at tyrosine 950 of the IGF-IR. These results indicate that in 32D cells, the IGF-IR regulates the expression of the Id2 gene and that this regulation is modulated by both positive and negative signals. Our results also suggest that in this model, Id2 proteins influence the differentiation program of cells but are not sufficient for the full stimulation of their proliferation program.

Download Full-text

Fibroblast Growth Factors Regulate Prolactin Transcription via an Atypical Rac-Dependent Signaling Pathway

Molecular Endocrinology ◽

10.1210/me.2003-0167 ◽

2003 ◽

Vol 17 (10) ◽

pp. 1921-1930 ◽

Cited By ~ 12

Author(s):

Twila A. Jackson ◽

David M. Koterwas ◽

Melissa A. Morgan ◽

Andrew P. Bradford

Keyword(s):

Gene Expression ◽

Growth Factors ◽

Signaling Pathway ◽

Promoter Activity ◽

Fibroblast Growth Factors ◽

Dominant Negative ◽

Pituitary Cells ◽

Dependent Manner ◽

Fibroblast Growth ◽

Prl Gene

Abstract Fibroblast growth factors (FGFs) play a critical role in pituitary development and in pituitary tumor formation and progression. We have previously characterized FGF signal transduction and regulation of the tissue-specific rat prolactin (rPRL) promoter in GH4 pituitary cells. FGF induction of rPRL transcription is independent of Ras, but mediated by a protein kinase C-δ (PKCδ)-dependent activation of MAPK (ERK). Here we demonstrate a functional role for the Rho family monomeric G protein, Rac1, in FGF regulation of PRL gene expression via an atypical signaling pathway. Expression of dominant negative Rac, but not RhoA or Cdc42, selectively inhibited FGF-induced rPRL promoter activity. Moreover, expression of dominant negative Rac also attenuated FGF-2 and FGF-4 stimulation of MAPK (ERK). However, in contrast to other Rac-dependent signaling pathways, FGF activation of rPRL promoter activity was independent of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase/Akt cascades. FGFs failed to activate JNK1 or JNK2, and expression of dominant negative JNK or Akt constructs did not block FGF-induced PRL transcription. Consistent with the role of PKCδ in FGF regulation of PRL gene expression, activation of the rPRL promoter was blocked by an inhibitor of phospholipase Cγ (PLCγ) activity. FGF treatment also induced rapid tyrosine phosphorylation of PLCγ in a Rac-dependent manner. These results suggest that FGF-2 and FGF-4 activate PRL gene expression via a novel Rac1, PLCγ, PKCδ, and ERK cascade, independent of phosphoinositol-3-kinase and JNK.

Download Full-text

Activation of Nuclear Factor κb and bcl-x Survival Gene Expression by Nerve Growth Factor Requires Tyrosine Phosphorylation of IκBα

The Journal of Cell Biology ◽

10.1083/jcb.152.4.753 ◽

2001 ◽

Vol 152 (4) ◽

pp. 753-764 ◽

Cited By ~ 100

Author(s):

Nguyen Truc Bui ◽

Antonia Livolsi ◽

Jean-Francois Peyron ◽

Jochen H.M. Prehn

Keyword(s):

Gene Expression ◽

Tyrosine Phosphorylation ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Nuclear Factor ◽

Human Neuroblastoma ◽

Tnf Α

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-κB (NFκB). We investigated the effect of NGF on the expression of Bcl-xL, an anti–apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1–10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFκB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFκB p65 subunit. NGF-induced NFκB activity and Bcl-xL expression were inhibited in cells overexpressing the NFκB inhibitor, IκBα. Unlike tumor necrosis factor-α (TNF-α), however, NGF-induced NFκB activation occurred without significant degradation of IκBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein–tagged IκBα. Moreover, in contrast to TNF-α, NGF failed to phosphorylate IκBα at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IκBα potently suppressed NFG-, but not TNF-α–induced NFκB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-α–, but not NGF-induced NFκB activation. We conclude that NGF and TNF-α induce different signaling pathways in neurons to activate NFκB and bcl-x gene expression.

Download Full-text

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

Journal of Cell Science ◽

10.1242/jcs.114.8.1579 ◽

2001 ◽

Vol 114 (8) ◽

pp. 1579-1589 ◽

Cited By ~ 2

Author(s):

M. Reyes-Reyes ◽

N. Mora ◽

A. Zentella ◽

C. Rosales

Keyword(s):

Signaling Pathways ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Small Gtpase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Monocytic Leukemia ◽

Monocytic Cells ◽

Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

Download Full-text

Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00279.2019 ◽

2020 ◽

Vol 318 (1) ◽

pp. C215-C224 ◽

Cited By ~ 1

Author(s):

Joaquin M. Muriel ◽

Andrea O’Neill ◽

Jaclyn P. Kerr ◽

Emily Kleinhans-Welte ◽

Richard M. Lovering ◽

...

Keyword(s):

Skeletal Muscle ◽

Isometric Force ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Type I ◽

Mrna Levels ◽

Force Transmission ◽

Keratin 18 ◽

Murine Skeletal Muscle

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout ( Krt18-KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Download Full-text

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line.

The Journal of Cell Biology ◽

10.1083/jcb.131.3.761 ◽

1995 ◽

Vol 131 (3) ◽

pp. 761-773 ◽

Cited By ~ 56

Author(s):

J M Rodier ◽

A M Vallés ◽

M Denoyelle ◽

J P Thiery ◽

B Boyer

Keyword(s):

Growth Factor ◽

Signaling Pathways ◽

Cell Line ◽

Intracellular Signaling ◽

Carcinoma Cell ◽

Biological Activities ◽

Dominant Negative ◽

Carcinoma Cell Line ◽

Dominant Negative Mutant ◽

Cell Scattering

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

Download Full-text

IGF1 suppresses cholesterol accumulation in the liver of growth hormone-deficient mice via the activation of ABCA1

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00134.2018 ◽

2018 ◽

Vol 315 (6) ◽

pp. E1232-E1241 ◽

Cited By ~ 1

Author(s):

Kensaku Fukunaga ◽

Hitomi Imachi ◽

Jingya Lyu ◽

Tao Dong ◽

Seisuke Sato ◽

...

Keyword(s):

Growth Hormone ◽

Specific Inhibitor ◽

Dominant Negative ◽

Luciferase Assay ◽

Dominant Negative Mutant ◽

Chip Assay ◽

Cholesterol Accumulation ◽

Adult Growth ◽

Abca1 Expression ◽

Deficient Mice

Recently, several clinical studies have suggested that adult growth hormone (GH) deficiency that also has low concentration of IGF1 is associated with an increased prevalence of fatty liver (FL). ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role on formation of FL. In this study, we determined the effects of IGF1 on ABCA1 expression in GH-deficient mice to clarify its effects on FL. Western blotting, real-time PCR, and a luciferase assay were employed to examine the effect of IGF1. The binding of FoxO1 to the ABCA1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Cholesterol accumulation was analyzed by Oil Red O stain and cholesterol content measurement. We confirmed that IGF1 upregulated the ABCA1 expression. The activity of a reporter construct containing the ABCA1 promoter was induced by IGF1, and this effect was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Constitutively active Akt stimulated the ABCA1 promoter activity, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element abolished the effect of IGF1. A ChIP assay indicated that FoxO1 mediated IGF1 transcriptional activity by directly binding to the ABCA1 promoter region. For in vivo experiments, we used an inhibitor for the GH receptor (Pegvisomant) to reduce the IGF1 level. A high-fat diet induced FL in mice (C57BL/6J) given Pegvisomant treatment. IGF1 treatment stimulated ABCA1 expression to improve cholesterol accumulation in these mice. These results show that the PI3K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF1 stimulation that suppressed FL in GH-deficient mice.

Download Full-text