PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells.
BACKGROUND
Axl is a receptor tyrosine kinase promoting anti-apoptosis, invasion and mitogenesis, and is highly expressed in different solid cancers. Axl basal transcriptional activity is driven by Sp1/Sp3, and overexpression of MZF-1 (myeloid zinc-finger 1) induces Axl transcription and gene expression. Furthermore, Axl expression is epigenetically controlled by CpG hypermethylation; however, little is known about inducible Axl gene expression and Axl regulation in haematopoetic malignancies.


RESULTS
In the present study, we studied Axl transcriptional regulation under PMA-stimulated conditions in leukaemia cells. Luciferase analysis with sequential 5'-deletion constructs revealed that the -660/-580 region of the Axl promoter is indispensable for induced promoter activity under PMA stimulation. This region includes AP-1 (activator protein 1)/CREB [CRE (cAMP-response-element)-binding protein] motifs, five times partially overlapping TGCGTG repeats and multiple GT repeats. Mutational, supershift and ChIP (chromatin immunoprecipitation) analysis determined that AP-1 family members bind to AP-1 motifs and to the 5 × TGCGTG overlapping repeats, thus transactivating Axl promoter activity. Furthermore, specific inhibitors of PKC (protein kinase C), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 reduced Axl expression. Additionally, mithramycin treatment abolished constitutive and PMA-induced Axl expression.


CONCLUSIONS
Taken together the results of the present study suggest that PMA-induced Axl gene expression in leukaemia cells is mediated by AP-1 motifs and 5 × TGCGTG repeats within the promoter region -660/-580, and through the PKC/ERK1/2/AP-1 or PKC/p-38/AP-1 signalling axis.
Department of Experimental Surgery Mannheim/Molecular Oncology of Solid Tumors, DKFZ and University of Heidelberg, Mannheim 68167, Germany.
Giridhar
G
Mudduluru
Jörg H
JH
Leupold
Philipp
P
Stroebel
Heike
H
Allgayer
D001483:Base Sequence; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*; D000094963:Axl Receptor Tyrosine Kinase
D011518:Proto-Oncogene Proteins; D018808:Transcription Factor AP-1; D020794:Receptor Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate; D000094963:Axl Receptor Tyrosine Kinase; C000722289:AXL protein, human