In Vitro and In Vivo Induction of Heme Oxygenase 1 in Mouse Macrophages following Melanocortin Receptor Activation

Abstract

RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4–24 h) and concentration-related (1–100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (Mφ) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary Mφ. HO-1 induction in peritoneal Mφ was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the Mφ, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.

A role for MC3R in modulating lung inflammation

“…We have previously shown that rodent knee joint and peritoneal macrophages express MC1R and MC3R [16,22] a cell type that responded with down-regulatory events to the addition of non-selective melanocortins. The expression of MC3R on MØ membrane protrusions was confirmed by immuno-gold labelling utilising electron microscopy [14].…”

“…To date five MCRs have been identified and they are all positively coupled to adenylate cyclase, signaling via protein kinase with agonism at these receptors leading to increases in intracellular cAMP [11]. Melanocortin peptides have long been reported to possess antiinflammatory effects in many experimental models of acute [12][13][14] and chronic inflammation, including inflammatory bowel disease [15], joint arthritis [16,17] and activation and consequent cytokine synthesis within the first few hours of treatment [20,21] and at later time points (>8h) induce the anti-inflammatory protein hemeoxygenase 1 [22] and IL-10 [24]. Only one study has so far addressed the role of α-MSH in modulating eosinophil accumulation levels in a murine model of allergic airway disease [19]: α-MSH treatment of allergic mice led to a significant reduction in inflammation and this protective effect was associated with augmented IL-10 production.…”

“…Following electrophorysis in a 10% polyacrylamide gel, membranes were blocked overnight in blocking solution containing 5% non-fat milk solution in Tris buffer saline containing 0.1% Tween-20 followed by anti-MC3R (1:2000) [22] synthesised by Dr Paolo Grieco as previously described [24,25], peptides were stored at -20°C prior to use, and dissolved in sterile PBS (pH 7.4).…”

The Gα12/13 Family of Heterotrimeric G Proteins and the Small GTPase RhoA Link the Kaposi Sarcoma-associated Herpes Virus G Protein-coupled Receptor to Heme Oxygenase-1 Expression and Tumorigenesis

“…Indeed, Rac1 is activated by the receptor and mediates vGPCR-induced transformation in cells and tumorigenesis in animal models (28,31), whereas RhoA is required for vGPCR-induced NFB activation and interleukin-8 secretion (29). Although it is known that some GPCRs induce HO-1 expression (5,(32)(33)(34), the nature of the molecules connecting these receptors to HO-1 expression has not been established.…”

“…Because of the antioxidant properties of the heme metabolism products, HO-1 has been considered a cytoprotector molecule involved in several physiological responses against inflammation and oxidative and cellular stress (2). However, an increasing number of studies have now expanded this notion defining HO-1 as an important regulator of the physiology of the vasculature, endothelial cell cycle control, proliferation, vascular endothelial growth factor (VEGF) 5 secretion, angiogenesis, and tumorigenesis (3, 8 -14). A recent study shows that HO-1 expression is induced in endothelial cells infected by the angiogenic and oncogenic Kaposi sarcoma-associated herpesvirus (KSHV) and that the enzyme is highly expressed in biopsy tissues from oral AIDS-Kaposi sarcoma lesions (15).…”