Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death.

Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis.

LPA-induced suppression of periostin in human osteosarcoma cells is mediated by the LPA(1)/Egr-1 axis.

Lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, mediates a multitude of (patho)physiological events including activation of mitogen-activated protein kinases (MAPKs). As LPA may induce cellular reponses in human osteosarcoma, the present study aimed at investigating expression of various LPA receptors, LPA-mediated activation of MAPK via G-protein coupling, and expression of early response genes in a cellular model for human osteosarcoma. We show that MG-63 cells express three members of the endothelial differentiation gene (Edg) family of G-protein coupled receptor transcripts (LPA(1-3)) but only two (LPA(4/5)) out of three members of the non-Edg family LPA receptor transcripts. Stimulation of MG-63 cells with LPA or synthetic LPA receptor agonists resulted in p42/44 MAPK phosphorylation via LPA(1)-LPA(3) receptors. Using pharmacological inhibitors, we show that LPA-mediated phosphorylation of p42/44 MAPK by LPA receptor engagement is transmitted by G(αi)-dependent pathways through the Src family of tyrosine kinases. As a consequence, a rapid and transient upregulation of the zinc finger transcription factor early growth response-1 (Egr-1) was observed. Egr-1 expression was strictly mediated via G(αi)/Src/p42/44 MAPK pathway; no involvement of the G(αq/11)/PLC/PKC or the PLD/PI3 kinase/Akt pathways was found. LPA-induced expression of functional Egr-1 in MG-63 cells could be confirmed by electrophoretic mobility shift assay. LPA-induced Egr-1 upregulation was accompanied by a time-dependent decrease of periostin (previously called osteoblast-specific factor 2), a cell adhesion protein for pre-osteoblasts. Silencing of LPA(1) and/or Egr-1 in MG-63 cells reversed LPA-mediated suppression of periostin. We here demonstrate a crosslink between Egr-1 and periostin in cancer cells, in particular in human osteosarcoma.

15-Deoxy-Δ12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species.

Prostaglandins (PGs), important modulators in bone biology, may also contribute to tumor formation and progression in human osteosarcoma. 15-Deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), a metabolite of PGD(2) and PPARγ-ligand, exerts a panel of biological activities via receptor-dependent and -independent mechanisms. As inducible cyclooxygenase-2 (Cox-2) is a candidate inflammatory marker in human osteosarcoma and a rate-limiting enzyme in PG biosynthesis, this study aimed at investigating intracellular redox status and signaling cascades leading to Cox-2 induction in human MG-63 osteosarcoma cells. 15d-PGJ(2) induced the accumulation of reactive oxygen species (ROS) that in turn may lead to upregulation of Cox-2 via two different routes in a PPARγ-independent manner. First, phosphorylation of p38 MAPK directly enhances Cox-2 expression by promoting mRNA stability. Second, 15d-PGJ(2) induces activation of epidermal growth factor receptors and downstream activation of Cox-2 via phosphorylation of p42/44 MAPK. Glutathione precursor molecules reversed enhanced ROS levels and Cox-2 expression. Functional activity of Cox-2 expression was tested by measurement of PGE(2) and PGF(2α). The synthetic compound 9,10-dihydro-15d-PGJ(2) lacking the α,ß-unsaturated carbonyl group in the cyclopentenone ring did not exhibit the cellular responses observed with 15d-PGJ(2). We conclude that the electrophilic carbon atom of 15d-PGJ(2) is responsible for alterations in intracellular redox status and Cox-2 expression.

Real-time PCR-based determination of gene copy numbers in Pichia pastoris.

Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here.