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Interaction between the serine/threonine protein phosphatase calcineurin and the NFAT family of transcription factors plays a critical regulatory role in the regulation of a variety of physiological functions.   Calcineurin inhibitors are widely used as drugs to suppress inflammation and immune response, including protecting organ transplant recipients from rejection. Unfortunately, these drugs produce cardiovascular side effects including hypertension.  These side effects have been shown to be related to modulation of nitric oxide and prostanoid pathways and can be explained by the critical role calcineurin plays in the transcription factor regulated expression of inflammatory enzymes such as eNOS and COX-2.

A recent paper by A. Garcia-Redondo et al. examined the role of Rcan1, a regulator of calcineurin, in the altered vascular function and mechanical properties observed in cardiovascular diseases.  Rcan1 has previously been clearly implicated in the development of several inflammatory diseases and is generally accepted as a potent negative regulator of inflammation.  However, the role Rcan1 plays in regulation of the cardiovascular system is less understood, so Rcan1-/- mice and matched Rcan1+/+ controls were compared to determine key functions of Rcan1.

Plasma levels of the prostanoid PGE2 were compared using Arbor Assays PGE2 Multi-format EIA Kit (K051-H) and found to be similar in both genotypes.  Vascular and aortic contractile responses were also compared.   Phenylephrine-induced contraction was greater in the aortic segments of Rcan1-/- mice, but was not increased in segments from smaller arteries.  Both removal of the endothelium and addition of the NOS-inhibitor L-NAME increased aortic segment contraction in the Rcan1+/+ mice to levels similar to those observed in the knockout mice. This suggests that reduced availability of nitric oxide may play a role in the increased baseline contractile response seen in the knockout mice.  However, aortic expression of eNOS, activation of Akt, and aortic NO levels (Arbor Assays NO Colorimetric Detection Kit, K023-H) were all shown to be similar in both genotypes. Addition of an antioxidant blocked phenylephrine response only in the Rcan1-/- mice so it’s possible that oxidative stress may be impacting NO availability in the knockout mice.

COX-2 derived prostanoids have also been shown to reduce NO effects and increase vasoconstriction response in pathological conditions, so COX-2 levels were also examined.   Both COX-2 mRNA and protein levels were found to be higher in aorta and vascular smooth muscle cells (VSMCs) from the knockout mice as compared to the controls.   Adenovirus-mediated re-expression of Rcan1 in the Rcan1-/- VSMCs decreased COX-2 to levels similar to those observed in the control mice, confirming that the increased COX-2 expression is a direct result of the absence of Rcan1.

Because COX-2 can be active by both Calcineurin/NFAT- and NFkB-dependent pathways, the authors next looked closer at NFkB.  The NFkB inhibitor parthenolide reduced phenylephrine response in the Ran1 knockout mice but not in the controls.  Furthermore, levels of phosphorylated p65 protein were higher in both aortic sections and VSMCs from the Rcan1-/- mice as compared to the controls, suggesting higher NFkB activity in the absence of Rcan1.

Ultimately, there is still more work to be done to fully understand the mechanisms through which Rcan1 influences the cardiovascular system.  This paper demonstrates that Rcan1 knockout mice have a baseline pro-inflammatory vascular profile as compared to Rcan1+/+ controls including; activation of the calcineurin/NFAT pathway, activation NFkB and its related pathways, increased expression and activity of COX-2, and increased cytokine levels.   There was also some preliminary evidence of a potential link to increased oxidative stress levels in the knockout mice, which will need to be investigated further.