Epidermal growth factor EGF has been shown to
Epidermal growth factor (EGF) has been shown to increase the 12S-lipoxygenase mRNA level by about two-fold in human epidermoid carcinoma A431 cells . This enzyme was shown to be of the platelet-type and associated with the microsomal fraction of the cells , . A requirement for EGF receptor tyrosine kinase for the induction was indicated using specific tyrosine kinase inhibitors . Luciferase promoter assays combined with site-directed mutagenesis indicated that two Sp1-binding sites at −158 and −123 bases upstream of the initiation codon were essential for gene expression of the 12S-lipoxygenase , . Overexpression of c-Fos increased the expression of 12S-lipoxygenase mRNA in A431 cells, and co-transfection with c-Fos and c-Jun expression vectors synergistically increased the promoter activity . Overexpression of Ha-ras also increased the promoter activity in a dose- and time-dependent manner, and enhanced the amount of c-Jun binding to Sp1 , . EGF was shown to increase time-dependently the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino terminal kinase (JNK) in A431 cells . It is of interest that cultured rat lenses and primary human lens epithelial cells require a 12-lipoxygenase metabolite for growth in response to EGF and insulin .
In addition, many other factors have been shown to induce the 12S-lipoxygenases. For example, transforming growth factor-α (TGF-α) and phorbol 12-myristate 13-acetate (PMA) increase the expression of 12S-lipoxygenase activity and mRNA in human epidermoid A431 cells , . Platelet-derived growth factor (PDGF) causes a significant increase in the formation of the 12-lipoxygenase product, and markedly increases the leukocyte-type 12S-lipoxygenase mRNA and protein in porcine aortic smooth muscle cells . In rat skin, bradykinin and platelet-activating factor (PAF) selectively stimulate 12S-lipoxygenase in a dose-dependent way, and hepoxilins are formed from arachidonic AMI5 inhibitor . Angiotensin II increased the formation of 12-HETE that can activate the mitogen-activated protein (MAP) kinase cascade in a Chinese hamster ovary fibroblast cell line that overexpresses the angiotensin II receptor , .
Nervous system The first report on the role of 12-lipoxygenase products in the neuronal system appeared in 1987 . The report showed that 12-HPETE was produced in Aplysia sensory neurons stimulated with a tetrapeptide (FMRFamide) and served as a second messenger for presynaptic inhibition. Application of 12-HPETE to the bath increased a presynaptic K+ conductance in Aplysia sensory cells. It was later reported that 12-HPETE could be replaced by the corresponding 12-keto acid or hepoxilin A3, both of which are metabolites of 12-HPETE , . The modulation of the small conductance K+ channel by the application of 12-HPETE onto the Aplysia neuron was also reported . In 1995, 12-HETE but not 12-HPETE was reported to increase M-type K+ currents in bullfrog neurons . A more recent report showed that 12-HETE was involved in μ-type opioid receptor inhibition by modulating a voltage-dependent K+ conductance in rat periaqueductal grey . It was also demonstrated that the angiotensin II receptor-mediated increase in the voltage-dependent delayed rectifier K+ current in neurons cultured from newborn rat hypothalamus and brain stems was partially mediated through 12-HETE . In these earlier studies there was inconsistent evidence for the involvement of 12-lipoxygenase products in producing small changes of K+ channels. What type of K+ channels is regulated in either a positive or negative way by 12-lipoxygenase products? The question arises because the earlier experiments were performed by classical pharmacological methods using non-specific 12-lipoxygenase inhibitors or 12-lipoxygenase products that themselves are unstable in aqueous solution. Furthermore, these products are very hydrophobic and difficult to dissolve completely at micromolar concentrations used in most experiments. To circumvent these technical problems, we attempted to overexpress human platelet 12S-lipoxygenase in neuroblastoma×glioma hybrid NG108-15 cells. These cells exhibit many properties of intact neurons such as the neurite extension, synapse formation with cultured muscle cells and acetylcholine release upon differentiation with agents that elevate cAMP concentrations . Differentiated NG108-15 cells become electrically excitable with increasing numbers of voltage- and Ca2+-gated ion channel molecules . We found that the membranes of 12S-lipoxygenase-expressing NG108-15 cells were much more excitable than mock-transfected or wild-type cells . A 1-s depolarizing current step of 0.32nA evoked more than 10 action potentials in the 12S-lipoxygenase-expressing cells, whereas the same stimulation evoked only two to three action potentials in wild-type or mock-transfected cells. We searched carefully for ion channels exhibited such a tremendous difference in excitability upon 12S-lipoxygenase overexpression. A voltage-dependent K+ channel generating M-current is one of the critical regulators of neuronal excitability . Among ion channels, only the K+ channel generating M-current was significantly inhibited in the many clones of the 12S-lipoxygenase-expressing cells examined to date using the whole cell variant of the patch–clamp technique used in the discontinuous voltage clamp mode. Furthermore, the cells treated with a 12-lipoxygenase inhibitor recovered action potential frequency and M-current amplitude to control levels. Other outward or inward currents were not affected in 12S-lipoxygenase-expressing cells, so far examined .