RESUMO
Transglutaminase 2 (TG2) is a multifunctional enzyme and two isoforms, TG2-L and TG2-S, exerting opposite effects in the regulation of cell death and survival, have been revealed in cancer tissues. Notably, in cancer cells a hypoxic environment may stimulate tumor growth, invasion and metastasis. Here we aimed to characterize the role of TG2 isoforms in neuroblastoma cell fate under hypoxic conditions. The mRNA levels of TG2 isoforms, hypoxia-inducible factor (HIF)-1α, p16, cyclin D1 and B1, as well as markers of cell proliferation/death, DNA damage, and cell cycle were examined in SH-SY5Y (non-MYCN-amplified) and IMR-32 (MYCN-amplified) neuroblastoma cells in hypoxia/reoxygenation conditions. The exposure to hypoxia induced the up-regulation of HIF-1α in both cell lines. Hypoxic conditions caused the up-regulation of TG2-S and the reduction of cell viability/proliferation associated with DNA damage in SH-SY5Y cells, while in IMR-32 did not produce DNA damage, and increased the levels of both TG2 isoforms and proliferation markers. Different cell response to hypoxia can be mediated by TG2 isoforms in function of MYCN amplification status. A better understanding of the role of TG2 isoforms in neuroblastoma may open new venues in a diagnostic and therapeutic perspective.
Assuntos
Proteínas de Ligação ao GTP/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Transglutaminases/genética , Ciclo Celular/genética , Morte Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Fragmentação do DNA , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Deposition of amyloid-beta (Aß) peptides has been shown to induce the release of inflammatory factors by activated microglia and brain infiltrating monocytes/macrophages. Interestingly, the enzyme transglutaminase 2 (TG2) has been shown to play a key role in neuroinflammation and regulation of transcription factors involved in immunomodulation. In this study, we aimed to better elucidate the mechanisms underlying TG2 involvement in the pro-inflammatory signaling pathway activated by fibrillar Aß1-42 in THP-1 monocytes. Cell exposure for 24 h to 500 nM Aß1-42, induced the up-regulation of CD14, CD16, and TG2, suggesting THP-1 cell functional activation. Aß1-42 also increased the production of reactive oxygen species, that was reduced by the pre-incubation with genistein (25 µg/ml), a soy isoflavone with antioxidant properties. Moreover, IL-1ß and IL-6 mRNA transcript and protein levels were eightfold increased in Aß1-42-treated THP-1 monocytes. Interestingly, these effects were significantly reduced by R283 (~45%), a specific inhibitor of TG activity, and genistein (~40%). Aß1-42 induced the activation of p54/p46 JNK, as well as ERK 1/2 at a lower extent. The inactivation of ERK1/2 signalling pathway, but not JNK, by either genistein or U0126, a MEK1/2 inhibitor, was not able to blunt Aß1-42-induced TG2 up-regulation, that, instead, was significantly reduced by R283. Aß1-42 also induced AP-1 activation that was not significantly affected by genistein or U0126, while was strongly reduced by R283. Our preliminary findings first suggest that TG2 up-regulation is involved in the pro-inflammatory activation of THP-1 monocytes induced by Aß1-42 via AP1/JNK signalling pathways.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Proteínas de Ligação ao GTP/genética , MAP Quinase Quinase 4/genética , Fragmentos de Peptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/genética , Transglutaminases/genética , Peptídeos beta-Amiloides/antagonistas & inibidores , Butadienos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Genisteína/farmacologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , MAP Quinase Quinase 4/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Proteína 2 Glutamina gama-Glutamiltransferase , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transdução de Sinais , Células THP-1 , Fator de Transcrição AP-1/metabolismo , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismoRESUMO
Recent evidence emphasized that transglutaminase 2 (TG2), a protein cross-linking enzyme, may play a role in the early phase of inflammation. High levels of TG2 have been associated with the constitutive activation of nuclear factor-kappa B (NF-κB) that is considered the main regulator of inflammation. In this context, the receptor activator of NF-kappa B ligand (RANKL) and receptor activator of NF-κB have extensive functions in the regulation of cytokine secretion associated with different pathological conditions. The human periodontal ligament (HPDL) cells, which express and secrete osteoprotegerin (OPG) and RANKL, represent an useful "ex vivo" model for monitoring cell response in inflammatory microenvironments, such as periodontitis-dependent tissue response. Thus, we evaluated TG2 expression and alterations in RANKL/OPG ratio occurring in cultured HPDL cells. The HPDL cells were obtained from patients with chronic periodontitis (CP) and healthy subjects. We observed the up-regulation of some inflammatory markers, such as IL-6, TNF-α, and HMGB-1, and at the same time an increase in TG2 mRNA levels in HPDL cells from CP patients compared with healthy subjects. We found a positive correlation between RANKL/OPG ratio and TG2 mRNA levels in HPDL cells from CP patients. In the parallel experiments, we demonstrated that TG2 inhibition reduced RANKL expression in both HPDL cells from CP patients and monocytes differentiated to macrophages by tetradecanoyl phorbol acetate treatment. Given the RANKL key role in NF-κB pathway and the observed up-regulation of pro-inflammatory cytokines, our data suggest that TG2 may be involved in molecular mechanisms of inflammatory response occurring in periodontal disease.