Multiple studies have shown EETs to be potent mitogens that activate EGF-R and ERKs in various cell types8, 10
. Previous studies by our group also demonstrated that EETs elevate expression and activity of MMPs in different human cancer cell lines9
. However, the role of EETs in EGF-R activation in human cancer cells remains unclear. In the present study, we found that addition of 14,15-EET or overexpression of a selective 14,15-EET epoxygenase can induce activation of EGF-R and ERK1/2 in multiple human derived cancer cell lines; Tca-8113, A549, HepG2 and MDA-MB-23. These signaling events are abolished by the tyrosine kinase inhibitor of EGF-R, AG1478. Interestingly, addition of 1,10-phenanthroline (a non-specific MMP inhibitor) or diphtheria toxin/CRM197 (an inhibitor of HB-EGF release) also blocked EET-induced activation of EGF-R and ERK1/2. As expected, inhibition of MMPs or HB-EGF cleavage did not block EGF induced EGF-R phosphorylation and its downstream activation of ERK1/2 in the cancer cells. Together, these results demonstrate that MMP activation, followed by HB-EGF cleavage and release, is essential for EET-induced EGF-R activation in human cancer cells.
HB-EGF is synthesized as a type I transmembrane protein, similar to other members of the epidermal growth factor (EGF) family. Pro-HB-EGF can be enzymatically shed within the juxtamembrane region to release a soluble 14–22 kDa growth factor14, 15
. The ectodomain shedding of pro-HB-EGF is induced by various stimuli such as phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)16, 17
, calcium ionophore18
, and various growth factors and cytokines19
. G-protein coupled receptor (GPCR) agonists also stimulate pro-HB-EGF shedding, which mediates EGF-R transactivation by GPCR signaling20, 21
. Metalloproteinases are responsible for the proteolytic cleavage of pro-HB-EGF since the ectodomain shedding of pro-HB-EGF is efficiently inhibited by various metalloproteinase inhibitors. Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase may be required for the activation of appropriate metalloproteinases since they are known to be involved in the intracellular signaling pathway for pro-HB-EGF shedding22, 23
In human colon carcinoma cells, IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage of pro-HB-EGF 24. Lysophosphatidic acid (LPA)-induced ectodomain shedding of pro-HB-EGF is critical for tumor formation in ovarian cancer25
. Deoxycholyltaurine (DCT)-induced transactivation of EGF-R is mediated by MMP-7-catalyzed release of the EGF-R ligand HB-EGF in H508 human colon cancer cells26
. In this study, we demonstrate that addition of 14,15-EET or overexpression of a 14,15-EET-specific epoxygenase leads to EGF-R transactivation via MMP activation and release of HB-EGF in four cancer cells. However, the identification of the specific metalloproteinase responsible for 14,15-EET-induced cleavage of the pro-HB-EGF requires further study.
Transactivation of EGF-R and the subsequent activation of downstream ERKs by metalloproteinase-mediated release of soluble HB-EGF play an important role in EET-stimulated mitogenic signaling. The tyrosine kinase inhibitor of EGF-R, AG1478, may provide significant therapeutic value in controlling the malignant growth of carcinomas. However, as demonstrated in previous studies, EETs activate the PI3K/Akt signaling pathway in different cell lines and tissues, which can also lead to mitogenic effects6, 8
. The relative importance of PI3K/Akt and EGF-R/ERK/MMP signaling pathways in mediating the mitogenic effects of CYP epoxygenase products remain to be determined.
In summary, this study reveals that induction of EGF-R transactivation is a crucial event in the mitogenic signaling transmission of EETs in cancer cells. In addition, EET-induced transactivation of EGF-R is mediated by activation of metalloproteinases that cleave pro-HB-EGF from the cell membrane and release active HB-EGF, which subsequently binds to EGF-R and activates downstream ERKs. Thus, CYP epoxygenase-derived EETs lead to malignant proliferation of cancer cells and growth of carcinomas via transactivation of EGF-R via a MMP-HB-EGF pathway. Further studies will be required to identify the precise metalloproteinases that are activated by EETs in cancer cells and to elucide the relative importance of EGF-R/ERK/MMP and other signaling pathways in mediating the mitogenic effects of CYP epoxygenase products.