Roaccutane Science :: How does the acne and cancer drug (Ro)accutane work chemically? :: Section 7: Gene-expression :: Significant inhibition of Sp1 phosphorylation Author Topic: Significant inhibition of Sp1 phosphorylation (Read 979 times) Max Administrator member is offline Joined: Apr 2005 Gender: Male Posts: 576 Significant inhibition of Sp1 phosphorylation « Thread Started on Jul 17, 2005, 7:51am » Gene-expression through Sp1 Modulation of Sp1 activity by nuclear receptors is a novel mechanism by which fat-soluble hormones regulate gene expression. RARs/RXRs physically interact with Sp1, potentiate Sp1 binding to the GC box motifs, and thus enhance transactivation of the urokinase promoter, which lacks a canonical RAR-responsive element/RXR-responsive element [1]. Nuclear receptors [retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D(3) receptor, peroxisome-proliferator-activated receptor and retinoic X receptor] induce an electrophoretic mobility increase of Sp1-GC-rich DNA complexes. Concomitantly, binding of Sp1 to the GC-box is enhanced [2]. In Ehrlich tumor cells, Sp1-DNA binding was inhibited by phosphatase exposure, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. Immunoprecipitation experiments revealed that Sp1 is mostly phosphorylated on serine residues [3, 7] with less than 5% on threonine and none on tyrosine residues [7]. (Ro)accutane induced significantly decreases of phosphorylation of Sp1 through multiple pathways, leading to a decreased transcription A list of kinases and phosphatases are known to be involved in Sp1 phosphorylation: DNA-PK, CDK2, ERK, PKC, PI3 kinase, PP1, PP2a and more, depending on cell line and function [7]. Experimental observations have found (Ro)accutane induced significant inhibition of at least three of these, however, it is here suggested that (Ro)accutane can inhibit ERK and possibly even PP1, PP2a and DNA-PK as well: 1) Inhibition of CDK2 (Ro)accutane is found to inhibit multiple of these kinaseses and phosphatases: In LCLs, RA-induced LCL accumulation in the G0/G1 phases correlated with the loss of the catalytic activity of all three G1-associated CDKs (CDK2, CDK4 and CDK6) and with increased levels of underphosphorylated pRb and, in some LCLs, p130. LCLs arrested in G0/G1 by RA also showed a significant decrease in the protein levels of cyclins D2, D3 and A, together with a reduction in the amount of cyclin D associated with CDK4 and CDK6, probably accounting for the inhibition of the relative kinase activity [5]. Cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. CDKs mediate phosphorylation of Sp1 [6]. 2) Inhibition of PKC Protein kinase C from 10T1/2 cells can be eluted by linear gradient of NaCl in two fractions. Following treatment with 10(-5) M 13-cis-retinoic acid a decrease of total PKC activity was observed [8]. 3) Inhibition of PI3K/Akt pathway In MTSV1-7 breast epithelial cells, CRBP-I inhibits, in a retinoic acid receptor-dependent manner, the PI3K/Akt survival pathway. Inhibition of PI3K/Akt was necessary and sufficient to explain the antitumor effects of CRBP-I and was mediated by decreased p85 regulatory and p110 catalytic subunit heterodimerization. We present evidence consistent with the idea that this effect is due to CRBP-I inhibition of p85 phosphorylation at Y688 [9]. Phosphatidylinositol 3-kinase (PI3K) is a critical signaling node that is regulated in response to the activation of growth factor receptors, G protein-coupled receptors, integrins and other cell signals that impact on cell survival and apoptosis. Class Ia PI3Ks consist of a regulatory (most commonly p85) and a catalytic (p110) subunit and catalyze the phosphorylation of phosphatidylinositol at the 3-hydroxy position of the inositide ring. The formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate is of particular relevance because PIP3 recruits downstream effectors to the plasma membrane. One such effector is Akt, which is activated by phosphoinositide-dependent kinase 1 which is likewise recruited to PIP3 sites. Akt is a serine−threonine protein kinase that once activated acts to inhibit apoptosis by phosphorylation of several key substrates. CRBP-I markedly inhibits Akt activity in cells in collagen I or in suspension relative to adherent monolayers, suggesting that Akt inhibition is a critical path through which CRBP-I promotes anoikis [9]. 4) Inhibition of ERK In HSC-1 cells, suppression of ERK1/2 and Akt activation is presumed to be involved in the RA-induced suppression of hTERT [10]. PPARgamma ligands In NSCLS cells, PPARgamma ligands also diminished the phosphorylation of cyclic adenosine monophosphate response element binding protein (CREB), diminished Sp1 nuclear protein expression, and prevented the binding of these transcription factors to CRE and Sp1 sites, respectively, within the Fn promoter. In summary, our results demonstrate that PPARgamma ligands inhibit Fn gene expression in NSCLC cells through PPARgamma-dependent and -independent pathways that affect both CREB and Sp1 [4]. Conclusion In small doses retinoic acid facilitate Sp1 binding to GC boxes (promoter regions). In toxic, supraphysiological doses retinoic acid is suggested to inhibit binding of Sp1 through decreased phosphorylation of Sp1 with at least two signal pathways involved, probably more. The inhibition of Sp1 phosphorylation is here suggested to span over all cell lines and regions where Sp1 promoter activity is essential. « Last Edit: Nov 3, 2005, 7:23am by Max » Link to Post - Back to Top   Logged Max Administrator member is offline Joined: Apr 2005 Gender: Male Posts: 576 Re: Sp1 and Sp3 « Reply #1 on Jul 19, 2005, 2:26pm » References: [1] Shimada J, Suzuki Y, Kim SJ, Wang PC, Matsumura M, Kojima S. Transactivation via RAR/RXR-Sp1 interaction: characterization of binding between Sp1 and GC box motif. (2001) Mol Endocrinol. Oct;15(10):1677-92. [2] Husmann M, Dragneva Y, Romahn E, Jehnichen P. Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation. (2000) Biochem J. Dec 15;352 Pt 3:763-72. [3] Segura JA, Donadio AC, Lobo C, Mates JM, Marquez J, Alonso FJ. Inhibition of glutaminase expression increases Sp1 phosphorylation and Sp1/Sp3 transcriptional activity in Ehrlich tumor cells. (2005) Cancer Lett. 2005 Jan 31;218(1):91-8. [4] Han SW, Ritzenthaler JD, Rivera HN, Roman J. Peroxisome proliferator-activated receptor gamma ligands suppress fibronectin gene expression in human lung carcinoma cells: Involvement of both cAMP response element (CRE) and Sp1. (2005) Am J Physiol Lung Cell Mol Physiol. May 20 [5] Zancai P, Cariati R, Rizzo S, Boiocchi M, Dolcetti R. Retinoic acid-mediated growth arrest of EBV-immortalized B lymphocytes is associated with multiple changes in G1 regulatory proteins: p27Kip1 up-regulation is a relevant early event. 1998 Oncogene. Oct 8;17(14):1827-36. [6] Fojas de Borja P, Collins NK, Du P, Azizkhan-Clifford J, Mudryj M. Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription. EMBO J. 2001 Oct 15;20(20):5737-47. [7] Chu S, Ferro TJ. Sp1: regulation of gene expression by phosphorylation. (2005) Gene. Mar 28;348:1-11. [8] Miloszewska J, Janik P, Pietruszewska E. Effect of 13-cis-retinoic acid treatment on protein kinase C from 10T1/2 cells. (1990) Folia Biol (Praha). 36(3-4):198-201. [9] Farias EF, Marzan C, Mira-y-Lopez R. Cellular retinol-binding protein-I inhibits PI3K/Akt signaling through a retinoic acid receptor-dependent mechanism that regulates p85-p110 heterodimerization. (2005) Oncogene. Feb 24;24(9):1598-606. [10] Kunisada M, Budiyanto A, Bito T, Nishigori C, Ueda M. Retinoic acid suppresses telomerase activity in HSC-1 human cutaneous squamous cell carcinoma. (2005) Br J Dermatol. Mar;152(3):435-43. « Last Edit: Jul 24, 2005, 9:33am by Max » Link to Post - Back to Top   Logged Max Administrator member is offline Joined: Apr 2005 Gender: Male Posts: 576 Re: Significant inhibition of Sp1 phosphorylation « Reply #2 on Jul 20, 2005, 8:06pm » Blood. 2005 Jul 1;106(1):304-10. Epub 2005 Mar 10. Related Articles, Links Arsenic suppresses gene expression in promyelocytic leukemia cells partly through Sp1 oxidation. Chou WC, Chen HY, Yu SL, Cheng L, Yang PC, Dang CV. Department of Laboratory Medicine, National Taiwan University Hospital, Taipei.wchou@ha.mc.ntu.edu.tw The mechanism by which arsenic dramatically affects gene expression remains poorly understood. Here we report that prolonged exposure of acute promyelocytic leukemia NB4 cells to low levels of arsenic trioxide increased the expression of a set of genes responsible for reactive oxygen species (ROS) production. We hypothesize that arsenic-induced ROS in turn contribute partially to altered gene expression. To identify genes responsive to arsenic-induced ROS, we used microarray gene expression analysis and identified genes that responded to arsenic and hydrogen peroxide but whose response to arsenic was reversed by an ROS scavenger, N-acetyl-L-cysteine. We found that 26% of the genes significantly responsive to arsenic might have been directly altered by ROS. We further explored the mechanisms by which ROS affects gene regulation and found that the Sp1 transcription factor was oxidized by arsenic treatment, with a corresponding decrease in its in situ binding on the promoters of 3 genes, hTERT, C17, and c-Myc, whose expressions were significantly suppressed. We conclude that ROS contributed partly to arsenic-mediated gene regulation and that Sp1 oxidation contributed to gene suppression by arsenic-induced ROS. PMID: 15761015 [PubMed - indexed for MEDLINE] Brain Res Mol Brain Res. 2003 Apr 10;112(1-2):153-62. Related Articles, Links Role of an AP-2-like element in transcriptional regulation of mouse micro-opioid receptor gene. Ko JL, Liu HC, Loh HH. Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. kojane@shu.edu Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse micro-opioid receptor (MOR) gene. In this study, we defined another functional element located the in -450 to -400 bp (translational start site designated as +1) region of the proximal promoter, which is also essential for the full promoter activity. It is designated as the morAP-2-like element for its sequence homologous to the consensus AP-2 element. Surprisingly, electrophoretic mobility shift analysis (EMSA) revealed that Sp1 and Sp3, but not AP-2 proteins, were specifically bound to the morAP-2-like element. Mutation of the morAP-2-like element, resulting in a loss of Sp binding, led to an approximately 35% decrease in activity, further confirming the positive role of the morAP-2-like element in MOR gene expression. Dephosphorylation of Sp proteins with alkaline phosphatase also decreased Sp binding to the morAP-2-like element in EMSA, suggesting phosphorylation of Sp is essential for its binding to this element. However, direct or indirect activation of PKA, a classical G-protein coupled signaling pathway, resulted in no significant change of Sp binding to the morAP-2-like element, nor of the promoter activity the SH-SY5Y cells, MOR expressing cells, suggesting that phosphorylation of Sp does not involve PKA. These results suggest that the binding of different phosphorylated forms of Sp proteins to the morAP-2-like element may contribute to the fine tuning of MOR expression in different cells. PMID: 12670713 [PubMed - indexed for MEDLINE] « Last Edit: Nov 3, 2005, 7:22am by Max » Link to Post - Back to Top   Logged Alexandre Guest Re: Significant inhibition of Sp1 phosphorylation « Reply #3 on Sept 30, 2006, 4:17pm » If you do not mind... IP3/Akt pathway is normally inhibited by PTEN. Downregulation of PTEN in melanoma cells promotes cancer. http://www.bioscience.org/2005/v10/af/1755/figures.htm Link to Post - Back to Top   Logged Forum Jump--------------------» Home--------------------» How does the acne and cancer drug (Ro)accutane work chemically?--------------------- Section 1: Significant irreversible hormonal antagonism- Section 2: Affected neurotransmitters and parts of the brain- Section 3: Programmed cell death (apoptosis)- Section 4: (Ro)accutane and the immune-defense- Section 5: (Side)-effects in subjects exposed to retinoic acid and their suggested causes (A-Z) --- Section 5b: Other long term effects- Section 6: (Ro)accutane effects suggested to worsen with age- Section 7: Gene-expression- Section 8: (Ro)accutane and its effects explained - for the beginner- Section 9: Los efectos de Roacutan, informacion en español- Section 10: Effekter inducerade av Roaccutan - Section 11: Information des effets a persuade de Roaccutane en français.- Section 12: Wirkungen von Roaccutan (isotretinoin) - Section 13: Gli effetti da Roaccutan- Section 14: Resultaten van Roaccutan--------------------» Discussion of treatments of (Ro)accutane (side)-effects----------------------------------------» Links--------------------- Links--------------------» Non commercial forum, no recommendation of marked products through banners----------------------------------------» General--------------------- General Board