Radiation-induced upregulation of γ-glutamyltransferase in colon carcinoma cells is mediated through the Ras signal transduction pathway

The activity of γ-glutamyltransferase (GGT) is frequently upregulated in tumor cells after oxidative stress and may thus increase the availability of amino acids needed for biosynthesis of the antioxidant glutathione. As γ-radiation of tumor cells can result in oxidative stress, we investigated whether such treatments modulate the enzyme level in colon carcinoma CC531 cells. Radiation of these cells blocked cell proliferation, increased cellular size, initiated apoptosis and upregulated GGT activity and protein levels in a dose- and time-related manner. A slight but significant increase in the cellular level of reactive oxygen species (ROS) was found directly after radiation but appeared not to cause the GGT elevation. Thus, other mechanisms than cellular oxidative stress appear to be responsible for the radiation-induced upregulation of GGT. Stable transfection of activated Ras in a human colon carcinoma cell line expressing wild-type Ras resulted in an increased GGT level, while a reduced enzyme level was demonstrated in another cell line with constitutively activated Ras after stably transfection with a dominant-negative Ras mutant. Moreover, addition of specific protein kinase inhibitors that blocked downstream targets PI-3K and MEK1/2 of Ras, prior to and after radiation, attenuated the radiation-induced activation of GGT. These results support a role for Ras, being frequently activated after radiation, in regulating the level of GGT and also indicate that GGT participates in radioresistance.     

Radiation-induced upregulation of gamma-glutamyltransferase in colon carcinoma cells is mediated through the Ras signal transduction pathway

The activity of gamma-glutamyltransferase (GGT) is frequently upregulated in tumor cells after oxidative stress and may thus increase the availability of amino acids needed for biosynthesis of the antioxidant glutathione. As gamma-radiation of tumor cells can result in oxidative stress, we investigated whether such treatments modulate the enzyme level in colon carcinoma CC531 cells. Radiation of these cells blocked cell proliferation, increased cellular size, initiated apoptosis and upregulated GGT activity and protein levels in a dose- and time-related manner. A slight but significant increase in the cellular level of reactive oxygen species (ROS) was found directly after radiation but appeared not to cause the GGT elevation. Thus, other mechanisms than cellular oxidative stress appear to be responsible for the radiation-induced upregulation of GGT. Stable transfection of activated Ras in a human colon carcinoma cell line expressing wild-type Ras resulted in an increased GGT level, while a reduced enzyme level was demonstrated in another cell line with constitutively activated Ras after stably transfection with a dominant-negative Ras mutant. Moreover, addition of specific protein kinase inhibitors that blocked downstream targets PI-3K and MEK1/2 of Ras, prior to and after radiation, attenuated the radiation-induced activation of GGT. These results support a role for Ras, being frequently activated after radiation, in regulating the level of GGT and also indicate that GGT participates in radioresistance.     

Regulation of beta-amyloid precursor protein expression by brain-derived neurotrophic factor involves activation of both the Ras and phosphatidylinositide 3-kinase signalling pathways

Brain-derived neurotrophic factor (BDNF) stimulates beta-amyloid precursor protein (APP) promoter activity by a Ras-dependent mechanism in TrkB-expressing SH-SY5Y cells. To determine the signalling pathways involved in the BDNF-induced response, we have analysed the ability of TrkB mutated forms to mediate promoter stimulation. Brain-derived neurotrophic factor causes a significant induction of promoter activity and mutation K540R in the active site of TrkB completely abolishes the neurotrophin-induced response. A substitution of the Y484 residue by phenylalanine, which blocks binding of Shc, reduces the activation of APP promoter by BDNF by approximately 50% whereas mutation Y785P, which blocks binding of phospholipase C gamma, does not affect the response. In addition, the phosphatidylinositide 3-kinase (PI3K)-specific inhibitors wortmannin and LY294002 reduced BDNF-induced activation. In agreement with a participation of both Ras/MAPK- and PI3K/Akt-mediated mechanisms, transient expression of constitutive active forms of Ras, PI3K and other components of both signalling pathways led to a significant increase of APP promoter activity. Furthermore, the stimulation of the APP promoter by BDNF was completely precluded by expression of dominant-negative forms of Ras and PI3K effectors. Taken together, our results suggest that simultaneous activation of at least two signalling pathways, Ras/MAPK and PI3K/Akt, is necessary to mediate a full activation of the APP promoter by BDNF.     

Expression of human cystatin A by keratinocytes is positively regulated via the Ras/MEKK1/MKK7/JNK signal transduction pathway but negatively regulated via the Ras/Raf-1/MEK1/ERK pathway.

Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form of ras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1, MEK1, ERK1, ERK2, or wild-type MEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1, ERK1, ERK2, or dominant negative forms of MEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (-272 to -278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, or p38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK.     

Regulation of calcium signalling by the small GTP-binding proteins Ras and Rac1

In order to investigate a possible interaction of the small GTP-binding proteins Ras and Rac1 with Ca²⁺-mediated signalling cascades the effects of dominant negative mutants of Ras and Rac1 on Ca²⁺ signalling have been studied after stimulation of either the EGFR or the nerve growth factor receptor (TRK). Expression of dominant negative Ras blocks the release of Ca²⁺ from internal stores after activation of EGFR whereas the calcium signal elicited by the activated TRK receptor is unaffected. The sensitivity to dominant negative Ras is determined by the structure of the PLCγ-binding sites of the corresponding receptors. Exchange of the PLCγ-binding domain of the EGFR by the PLCγ-binding site of TRK renders the EGFR-induced calcium signal insensitive to the expression of dominant negative Ras. Substitution of the PLCγ-binding site of TRK by the PLCγ-binding region of EGFR renders TRK sensitive to dominant negative Ras. The inhibition of Ca²⁺ release by dominant negative Ras is accompanied by a reduction in PLCγ binding to the EGFR and a concomitant decrease of EGF-induced inositol-1,3,5-trisphosphate (InsP₃) formation. The depression of PLCγ binding to EGFR is explained by a competition of PLCγ with other SH2-domain containing proteins for the same low affinity binding regions of the EGFR. This conclusion is supported by the observation that microinjection of several SH2-domain containing proteins including Ras-GP, lipase-free fragment of PLCγ or Janus kinase binding protein (JAB), reduces the association of PLCγ to the EGFR, not, however, to TRK. In contrast to dominant negative Ras which does not affect the Ca²⁺ transient induced by the activation of the TRK receptor, a dominant negative mutant of Rac significantly depresses the Ca²⁺ signals induced by EGFR as well as by TRK. The different behavior of Rac and Ras supports the notion that the two small GTP-binding proteins act through separate pathways. It is demonstrated that dominant negative Rac significantly reduces the formation of phosphatidylinositol-4,5-bisphosphate (PIP₂), the substrate of PLCγ. This effect is not observed after expression of dominant negative Ras. In summary, the data provide further evidence for a cross-talk between small GTP-binding proteins and Ca²⁺ signalling in which both G-proteins interfere with the formation of InsP₃ although by different mechanisms.     

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

Abstract

γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.