Roles of Rac and cytosolic phospholipase A2 in the intracellular signalling in response to titanium particles

Titanium (Ti) particle is one of the prosthetic materials commonly used in implantation and has frequently been implicated in pathogenesis such as periprosthetic osteolysis. In the present study, we undertook to understand the intracellular signalling pathway stimulated by exogenous Ti at Rat-2 fibroblasts. By reporter gene analysis following transient transfections, exogenous Ti was shown to stimulate c-fos serum response element (SRE)-dependent luciferase activities in a dose-dependent manner. In addition, Ti-induced SRE activation was shown to be dramatically repressed by RacN17, a dominant negative mutant of Rac1, suggesting that Rac GTPase is essential for the signalling of Ti to c-fos SRE. Furthermore, pretreatment with MAFP, an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), MK886, an inhibitor of 5-lipoxygenase (5-LO), or indomethacin, a general inhibitor of cyclooxygenase (COX), also significantly repressed Ti-induced SRE activation, suggesting mediatory roles of cPLA(2) and subsequent arachidonic acid (AA) metabolisms to leukotrienes (LTs) and prostaglandins (PGs) in the Ti signalling to c-fos SRE. Consistent with these results, intracellular levels of leukotriene B(4) (LTB(4)) and prostaglandin E(2) (PGE(2)) were Rac-dependently elevated in cells exposed to Ti particles.     

G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.     

Role of the cytosolic phospholipase A2-linked cascade in signaling by an oncogenic, constitutively active Ha-Ras isoform

Activation of Ras signaling by growth factors has been associated with gene regulation and cell proliferation. Here we characterize the contributory role of cytosolic phospholipase A(2) in the oncogenic Ha-Ras(V12) signaling pathway leading to activation of c-fos serum response element (SRE) and transformation in Rat-2 fibroblasts. Using a c-fos SRE-luciferase reporter gene, we showed that the transactivation of SRE by Ha-Ras(V12) is mainly via a Rac-linked cascade, although the Raf-mitogen-activated protein kinase cascade is required for full activation. In addition, Ha-Ras(V12)-induced DNA synthesis was significantly attenuated by microinjection of recombinant Rac(N17), a dominant negative mutant of Rac1. To identify the mediators downstream of Rac in the Ha-Ras(V12) signaling, we investigated the involvement of cytosolic phospholipase A(2). Oncogenic Ha-Ras(V12)-induced SRE activation was significantly inhibited by either pretreatment with mepacrine, a phospholipase A(2) inhibitor, or cotransfection with the antisense oligonucleotide of cytosolic phospholipase A(2). We also found cytosolic phospholipase A(2) to be situated downstream of Ha-Ras(V12) in a signal pathway leading to transformation. Together, these results are indicative of mediatory roles of Rac and cytosolic phospholipase A(2) in the signaling pathway by which Ha-Ras(V12) transactivates c-fos SRE and transformation. Our findings point to cytosolic phospholipase A(2) as a novel potential target for suppressing oncogenic Ha-Ras(V12) signaling in the cell.     

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

Sequences at the 3' side of the c-fos SRE mediate gene expression via an Sob1-dependent, TCF-independent pathway.

We previously described a 110-kDa tyrosine phosphoprotein, Sob 1, that regulates formation of the DNA binding complex Band A at the c-fos serum response element (SRE) during T cell activation. Using competition and mutant oligonucleotide analysis, we have determined that both the core CArG box of the c-fos SRE and the 3' sequences flanking the CArG box are necessary for stable Band A complex formation. Moreover, using transient transfection and reporter assays, we show that mutations affecting Band A complex formation in vitro also impaired serum induction of c-fos gene expression in vivo. Since mutation at this site has no effect on SRF binding, our results suggest that in combination with SRE/SRF, Sob 1-regulated factor(s) bind at the 3' side of SRE to form Band A, and this confers maximal serum induction of c-fos gene expression via the SRE.     

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.