Kaposi’s sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi’s sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades. We show that vGPCR expression triggers signaling pathways that upregulate COX-2 levels due to a dual effect upon both its gene promoter region and, in mature mRNA, the 3’UTR region that control mRNA stability. Both events are mediated by signaling through ERK1/2 MAPK pathway. Inhibition of COX-2 in vGPCR-transformed cells impairs vGPCR-driven angiogenesis and treatment with the COX-2-selective inhibitory drug Celecoxib produces a significant decrease in tumor growth, pointing to COX-2 activity as critical for vGPCR oncogenicity in vivo and indicating that COX-2-mediated angiogenesis could play a role in KS tumorigenesis. These results, along with the overexpression of COX-2 in KS lesions, define COX-2 as a potential target for the prevention and treatment of KSHV-oncogenesis. Kaposi sarcoma (KS) is the most frequent AIDS-related cancer (AIDS-KS). The tumors originate in cells infected with a cancer-causing virus (KSHV). A gene encoded by the virus expresses a protein with oncogenic potential in the infected cells (vGPCR), which has the ability to promote cell transformation and angiogenesis driving KS tumorigenesis. Therefore, the identification of oncogenic intracellular signalling mechanisms triggered by vGPCR could be of therapeutic interest. Cyclooxygenase-2 (COX-2) is an inflammatory molecule involved in tumor angiogenesis that can be targeted by several FDA-approved non-steroidal anti-inflammatory drugs and specific inhibitors. Here, we demonstrate that vGPCR upregulates COX-2 activity and expression through upregulation of transcription and mRNA stability via an ERK1/2 dependent pathway. We also show that COX-2 activity is critical for vGPCR angiogenesis and oncogenesis using KSHV infection and mouse models. Consistent with a role in KS pathogenesis, we found that vGPCR upregulates COX-2 activity in endothelial cells, that it is essential for VEGF upregulation via vGPCR, and that it is expressed in KSHV infected cells of AIDS-KS lesions. These facts point to COX-2 as one of the molecular components of the vGPCR angiogenic switch in Kaposi Sarcoma and a potential target for chemoprevention and therapy.

The present study was designed to investigate the effect of lipopolysaccharide (LPS) on the expression levels and activities of the nitric oxide synthase (NOS) and heme oxygenase (HO) systems in the rat adrenal gland. Both enzymatic activities were significantly increased in this tissue after in vivo treatment with LPS. The concurrent induction of the HO-1, NOS-1, and NOS-2 gene products was also detected as both mRNAs and protein levels were augmented by this treatment in a time-dependent way. A significant interaction between both signaling systems was also demonstrated as in vivo blockage of NOS activity with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in HO expression and activity levels, while an increase in NOS activity was observed when HO was inhibited by Sn-protoporphyrin IX (Sn-PPIX). As both NOS and HO activities have been previously involved in the modulation of adrenal steroidogenesis, we investigated the participation of these signaling systems in the adrenal response to LPS. Our results showed that acute stimulation of steroid production by ACTH was significantly increased when either NOS or HO activities were inhibited. We conclude that adrenal NOS and HO can be induced by a non-lethal dose of endotoxin supporting a modulatory role for these activities in the adrenal response to immune challenges.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H₂O₂/Fe²⁺. ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at −146 and −37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[−295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[−295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of theHmox1gene.

While physiological levels of glucocorticoids are required to ensure proper functions of the body, consistently high levels may engender several deleterious consequences. We have previously shown an increase in the activity of the hypothalamic‐pituitary‐adrenal (HPA) axis in rats fed sucrose‐rich diets (SRD). The main goal of this study was to analyze the processes involved in the modulation of the pituitary production of ACTH by SRD, and to test melatonin as a possible therapeutic agent for the prevention of the HPA axis dysfunction. Male Wistar rats were fed standard chow and either SRD (30% sucrose in the drinking water) or plain water for three weeks. Melatonin was administered as subcutaneous pellets. Results showed that SRD treatment induced an increase in systemic ACTH and corticosterone levels and a decrease in melatonin levels. In the pituitary gland, we also detected an increase in the expression levels of proopiomelanocortin (POMC) that was accompanied by increased levels of: lipoperoxides, nitro‐tyrosine modified proteins, catalase, heme oxygenase‐1, interleukin‐1β mRNA, and by an increase in the tissue number of inflammatory cells (F4/80 and Iba‐1 positive cells). Melatonin treatment prevented all these systemic and pituitary changes as well as the increase in POMC expression induced by incubation of AtT‐20 corticotrophs with conditioned media obtained from stimulated macrophages. In conclusion, stimulation of POMC/ACTH production in rats fed a SRD could involve the generation of oxidative stress and inflammation in the pituitary gland. Melatonin treatment prevented these effects and normalized the activity of the HPA axis.