Abstract
SOCS-3 gene induction by cAMP-elevating agents or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in primary HUVECs was found to require PKCη- and PKCε-dependent extracellular signal-regulated kinase (ERK) activation. The minimal, ERK-responsive element of the SOCS-3 promoter was localized to a region spanning nucleotides −107 to the transcription start site and contains conserved binding sites for AP-1 and SP1/SP3 transcription factors, as well as proximal and distal signal transducer and activator of transcription (pSTAT and dSTAT) binding elements. All three classes of transcription factor were activated in response to ERK activation. Moreover, representative protein components of each of these transcription factor binding sites, namely c-Jun, STAT3, and SP3, were found to undergo ERK-dependent phosphorylation within their respective transactivation domains. Mutational analysis demonstrated an absolute requirement for the SP1/SP3 binding element in controlling basal transcriptional activity of the minimal SOCS-3 promoter. In addition AP-1, pSTAT, and SP1/SP3 binding sites were required for ERK-dependent, PMA-stimulated SOCS-3 gene activation. The dSTAT site seems to be important for supporting activity of the AP-1 site, because combined deletion of both sites completely blocks transcriptional activation of SOCS-3 by PMA. Together these results describe novel, ERK-dependent regulation of transcriptional activity that requires codependent activation of multiple transcription factors within the same region of the SOCS-3 gene promoter.
Footnotes
This work was supported by the British Heart Foundation [Grants PG/10/026/28303, PG/08/125/26415].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- C/EBP
- CCAAT/enhancer-binding protein
- cPKC
- conventional PKC
- CRE
- cAMP response element
- CREB
- cAMP response element-binding protein
- DAG
- diacyl glycerol
- EPAC
- exchange protein activated by cAMP
- ERK
- extracellular signal regulated kinase
- F/R
- forskolin/rolipram
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- Gö6983
- 3-[1-(3-dimethylamino-propyl)-5-methoxy-1H-indol-3-yl]4-(1H-indol-3-yl)pyrrolidine-2,5-dione
- HUVEC
- human umbilical vascular endothelial cell
- IL
- interleukin
- JAK
- Janus tyrosine kinase
- JNK
- c-Jun N-terminal kinase
- MAP
- mitogen-activated protein kinase
- MEK
- mitogen activated protein kinase kinase
- MG132
- N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
- MSH
- melanocyte stimulating hormone
- nPKC
- novel PKC
- PCR
- polymerase chain reaction
- PGE2
- prostaglandin E2
- AP-1
- activator protein 1
- PKC
- protein kinase C
- PMA
- phorbol 12-myristate 13-acetate
- Ro 31-7549
- bisindolylmaleimide VIII acetate
- RT
- reverse transcriptase
- SH2
- Src homology 2
- siRNA
- small interfering RNA
- SOCS
- suppressors of cytokine signaling
- SP
- specificity protein
- SP600125
- 1,9-pyrazoloanthrone
- STAT
- signal transducers and activators of transcription
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene.
- Received November 28, 2011.
- Accepted February 2, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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