Abstract
Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3′,5′-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.
Highlights
Gravity (g) alterations are known to affect gene expression and cellular functions
The present results indicate that hypergravity and simulated microgravity inversely modulate the expression of guanylyl cyclases (GC)-cGMP signaling components in human melanoma cells
In pigmented 1F6 melanoma cells, hypergravity (5 g for 24 h) induces an increase in the mRNA expression of Endothelial nitric oxide synthase (eNOS) and multidrug resistanceassociated proteins 4 (MRP4)/Multidrug resistance-associated protein (MRP5), but there are no apparent changes in the mRNA expression of Guanylyl cyclase A (GC-A)/Guanylyl cyclase B (GC-B) when compared to 1-g controls
Summary
Gravity (g) alterations are known to affect gene expression and cellular functions. Both, the condition of microgravity that means without the impact of the gravitational stimulus as well as hypergravity, which means increase of the gravitational stimulus to a moderate physiological level, have been proven as tools to elucidate the effects of gravity on Microgravity Sci. Cyclic GMP is an intracellular signaling molecule that regulates essential (patho)physiologigal processes in multiple cell types including tumor cells, such as smooth muscle relaxation, platelet aggregation, neurotransmission, cell growth and survival (Lucas et al 2000; Beavo and Brunton 2002; Bian et al 2012; Fajardo et al 2014) It can be generated from GTP by two classes of guanylyl cyclases (GC), the nitric oxide (NO)-sensitive soluble GC (sGC) (Derbyshire and Marletta 2012) or the peptide-activated membrane-bound GC, such as the atrial natriuretic peptide (ANP)-responsive GC-A or the C-type natriuretic peptide (CNP)-responsible GC-B (Potter et al 2006). Whereas MRP5 has much higher affinity to export cGMP than MRP4 (Sager 2004; Jedlitschky et al 2000; Wielinga et al 2003), both MRP4 and MRP5 export nucleoside analogs that are used in antiviral and anticancer therapy (Deeley et al 2006)
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