Faith B. Davis

The anti-inflammatory effects of selenium are mediated through 15-Deoxy-Δ12, 14-prostaglandin J2 in macrophages

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-κB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-κB cascade, IκB-kinase β (IKKβ) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKβ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKβ activity. Further analysis revealed that inhibition of IKKβ activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKβ, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-γ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium.

Acute cellular actions of thyroid hormone and myocardial function

The mechanisms of actions of thyroid hormone in various tissues are largely viewed as cell nucleus-mediated. However, several actions of this hormone are definitively extranuclear, and these include effects on the activities of Ca(2+)-adenosine triphosphatases (ATPases) of myocardial sarcolemma and, apparently, sarcoplasmic reticulum in animal models. Both effects would serve to reduce cytoplasmic (sarcoplasmic) [Ca2+]. Sarcoplasmic reticulum uptake of Ca2+ from sarcoplasm is mediated by Ca(2+)-ATPase and is deficient in end-stage heart failure; thyroid hormone can enhance sarcoplasmic reticulum Ca(2+)-ATPase activity acutely via an extranuclear mechanism or indirectly via the myosin-associated Ca(2+)-ATPase gene. Such actions would serve to improve myocardial relaxation, thus improvement in diastolic dysfunction, and may be cardioprotective if excessive levels of sarcoplasmic [Ca2+] develop during reperfusion of previously ischemic tissue. Action of thyroid hormone on sarcolemmal Ca(2+)-ATPase activity will enhance Ca2+ efflux, and a recently described effect of the hormone on myocardial Na+ inactivation current may serve to increase or reduce sarcoplasmic [Ca2+], depending upon the vector of Na+/Ca2+ exchange. This article reviews acute effects of thyroid hormone on the heart that are extranuclear in mechanism.

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca2+-ATPase

Membrane Ca(2+)-ATPase activity was stimulated in vitro separately by T4 (10(-10) M) and by epinephrine (10(-6) M). In the presence of a fixed concentration of T4, additions of 10(-8) and 10(-6) M epinephrine reduced the T4 effect on the enzyme. beta-Adrenergic blockade with propranolol (10(-6) M) prevented stimulation by epinephrine of Ca(2+)-ATPase activity, but did not prevent the suppressive action of epinephrine on T4-stimulable Ca(2+)-ATPase. In contrast, alpha 1-adrenergic blockade with unlabelled prazosin restored the effect of T4 on Ca(2+)-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulation by T4 of red cell Ca(2+)-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (Kd, 1.2 x 10(-8) M; Bmax, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains alpha 1-adrenergic receptor sites that are capable of regulating Ca(2+)-ATPase activity.

Differential induction of tumor necrosis factor α and manganese superoxide dismutase by endotoxin in human monocytes: Role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor κB†

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor α (TNFα) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFα by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen‐activated protein kinase (MAPK) pathway plays an important role in the LPS‐induced TNFα production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFα in human monocytes. Genistein (10 μg/ml) and herbimycin A (1 μg/ml) markedly inhibited LPS‐induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal‐regulated kinase), and increases in the steady state level of TNFα mRNA as well as TNFα production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS‐induced activation of nuclear factor κB (NFκB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFκB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFα and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFα, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFκB alone is insufficient for the induction of TNFα mRNA by LPS, it is necessary for the induction of TNFα as well as MnSOD mRNAs. J. Cell. Physiol. 182:381–389, 2000.

Specific inositol phosphates inhibit basal and calmodulin-stimulated Ca(2+)-ATPase activity in human erythrocyte membranes in vitro and inhibit binding of calmodulin to membranes.

d‐Myo‐inositol 1,4,5‐trisphosphate (Ins[1,4‐,5]P3) inhibits rat heart sarcolemmal Ca2+‐ATPase activity (T. H. Kuo, Biochem. Biophys. Res. Commun. 152: 1111, 1988). We have studied the effect and mechanism of action of Ins(1,4,5)P3 and related inositol phosphates on human red cell membrane Ca2+‐ATPase (EC 3.6.1.3) activity in vitro. At 10–6 m, Ins(1,4,5)P3 and d‐myo‐inositol 4,5‐bisphosphate (Ins[4,5]P2) inhibited human erythrocyte membrane Ca2+‐ATPase activity in vitro by 42 and 31%, respectively. d‐Myo‐inositol 1,3,4,5‐tetrakisphosphate, d‐myo‐inositol 1,4‐bisphosphate, and d‐myo‐inositol 1‐phosphate were not inhibitory. Enzyme inhibition by Ins(1,4,5)P3 was blocked by heparin. Exogenous purified calmodulin also stimulated red cell membrane Ca2+‐ATPase activity; this stimulation was inhibited by Ins(1,4,5)P3. Ins(4,5)P2 and Ins(1,4,5)P3, but not Ins(1,4)P2, inhibited the binding of [125I]calmodulin to red cell membranes. Thus, specific inositol phosphates reduce plasma membrane Ca2+‐ATPase activity and enhancement of the latter in vitro by purified calmodulin. The mechanism of these effects may in part relate to inhibition by inositol phosphates of binding of calmodulin to erythrocyte membranes.—Davis, F. B.; Davis, P. J.; Lawrence, W. D.; Blas, S. D. Specific inositol phosphates inhibit basal and calmodulin‐stimulated Ca2+‐ATPase activity in human erythrocyte membranes in vitro and inhibit binding of calmodulin to membranes. FASEB J. 5: 2992‐2995; 1991.

Thyroid hormone analogues potentiate the antiviral action of interferon‐γ by two mechanisms

L‐thyroxine (L‐T4) potentiates the antiviral activity of human interferon‐γ (IFN‐γ) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN‐γ (1.0 IU/ml), 2) for 24 h cotreatment with IFN‐γ, 3) for 4 h, after 20 h cell incubation with IFN‐γ, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN‐γ. The antiviral effect of IFN‐γ was then assayed. L‐T4 potentiated the antiviral action of IFN‐γ by a reduction in virus yield of more than two logs, the equivalent of a more than 100‐fold potentiation of the IFNs antiviral effect. 3,3′,5‐L‐triiodothyronine (L‐T3) was as effective as L‐T4 when coincubated for 24 h with IFN‐γ but was less effective than L‐T4 when coincubated for only 4 h. D‐T4, D‐T3, 3,3′,5‐triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5‐diiodothyronine (T2) were inactive. When preincubated with L‐T4 for 24 h prior to IFN‐γ treatment, tetrac blocked L‐T4 potentiation, but, when coincubated with L‐T4 for 4 h after 20 h IFN‐γ, tetrac did not inhibit the L‐T4 effect. 3,3′,5′‐L‐triiodothyronine (rT3) also potentiated the antiviral action of IFN‐γ, but only in the preincubation model. Furthermore, the effects of rT3 preincubation and L‐T3 coincubation were additive, resulting in 100‐fold potentiation of the IFN‐γ effect. When L‐T4, L‐T3, or rT3, plus cycloheximide (5 μg/ml), was added to cells for 24 h and then removed prior to 24 h IFN‐γ exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN‐γ potentiation by 4 h of L‐T4 or L‐T3 coincubation was not inhibited by cycloheximide (25 μg/ml). These studies demonstrate two mechanisms by which thyroid hormone can potentiate IFN‐γs effect: 1) a protein synthesis‐dependent mechanism evidenced by enhancement of IFN‐γs antiviral action by L‐T4, L‐T3, or rT3 preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis‐independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L‐T4 or L‐T3, but not rT3, with IFN‐γ. The protein synthesis‐dependent pathway is responsive to rT3, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN‐γ can be regulated has not previously been described.