Tetyana V. Pedchenko

Oestrogen regulates sympathetic neurite outgrowth by modulating brain derived neurotrophic factor synthesis and release by the rodent uterus

Sympathetic innervation of the adult rodent uterus undergoes cyclic remodelling. Terminal sympathetic axons degenerate when oestrogen levels rise and regenerate when oestrogen levels decline. This study examined the role of neurotrophins in oestrogen‐mediated uterine sympathetic nerve remodelling. Oestrogen injection of ovariectomized female rats did not affect uterine NT‐3 levels 24 h postinjection, and increased endometrial NGF protein, indicating that reduced NGF or NT‐3 is not responsible for the oestrogen‐induced denervation. Oestrogen also raised BDNF protein and mRNA in myometrium and endometrium. To assess whether increased BDNF affects uterine receptivity to sympathetic outgrowth, sympathetic ganglion explants were co‐cultured with myometrium. Myometrium from ovariectomized rats induced neuritogenesis in oestrogen‐free conditions, and this was abolished when BDNF was added to the medium. Neuritogenesis induced by ovariectomized myometrium was suppressed by oestrogen, and restored by a BDNF function‐blocking antibody. To determine if target BDNF synthesis is required for oestrogen to suppress sympathetic neurite outgrowth, uteri from wild‐type mice and mice homozygous or heterozygous for recombinant mutations of the BDNF gene were cultured with rat sympathetic ganglia. Neuritogenesis induced by wild‐type uteri was diminished by oestrogen. Neurite formation in the presence of homozygous BDNF mutant uteri was not affected by oestrogen, but was lower than that of wild‐type mice. Uteri from mice heterozygous for the BDNF mutation, who have reduced BDNF synthesis, showed normal neuritogenic properties, but were not affected by oestrogen. These findings suggest that oestrogen alters neuritogenic properties of the rodent uterus by regulating BDNF synthesis, which inhibits sympathetic neurite outgrowth.

L-cycloserine slows the clinical and pathological course in mice with globoid cell leukodystrophy (twitcher mice).

Globoid cell leukodystrophy (Krabbes disease) is an autosomal recessive disease that affects the lysosomal enzyme galactosylceramidase. Galactosylceramidase removes galactose from galactosylceramide and psychosine, which are derived from sphingosine. In the present study, L‐cycloserine (an inhibitor of 3‐ketodyhydrosphingosine synthase) was administered to the twitcher mouse, an authentic model of globoid cell leukodystrophy. Twitcher mice treated with L‐cycloserine had a significantly longer life span and a delayed onset of weight loss than vehicle‐injected twitcher mice. Pathological features such as macrophage infiltration and astrocyte gliosis also were less in treated twitcher mice. These results indicate that substrate reduction therapy may have therapeutic value for individuals with residual enzymatic activity, e.g., individuals with late onset disease or individuals with partial enzyme replacement via bone marrow transplantation. In these cases, a reduction in galactosylceramide and psychosine synthesis would enable residual enzymatic activity to keep up with the accumulation of these substrates that would otherwise lead to pathology. J. Neurosci. Res. 60:231–236, 2000

NIK Is Involved in Nucleosomal Regulation by Enhancing Histone H3 Phosphorylation by IKKα

The exact physiological role of NF-κB-inducing kinase (NIK) in the NF-κB activation pathway has not been defined, although it is an upstream kinase of IKKα. Recent studies have indicated that IKKα is a nucleosomal modifier of NF-κB signaling. We hypothesized that NIK generates a proximal signal that contributes to IKKα modification of nucleosomal structure through phosphorylation of histone H3 and enhancement of target gene expression. By using a chromatin immunoprecipitation assay, our data show that endogenous IKKα is recruited to the promoter site of several NF-κB-dependent genes in macrophages. Our data show that immunoreactive NIK is rapidly recruited to nuclear compartment in macrophages in response to treatment with endotoxin where it augments phosphorylation of histone H3 by inducing phosphorylation and kinase activity of IKKα. A small interfering RNA knockdown of NIK markedly reduces phosphorylation of histone H3 in endotoxin treated macrophages. These data, together, demonstrate a novel role for NIK as a histone H3 modifier, through an accessory pathway from NIK to IKKα, that could play an important role in the endotoxin response through modification of nucleosomal structure.

IL-6 deficiency causes enhanced pathology in Twitcher (globoid cell leukodystrophy) mice.

The expression of IL-6 is greatly enhanced in the twitcher mouse (S. M. LeVine and D. C. Brown, 1997, J. Neuroimmunol. 73, 47-56), which is an authentic animal model of globoid cell leukodystrophy (Krabbes disease). In order to investigate the role of IL-6 in this disease, twitcher/IL-6-deficient mice were generated and the pathology was compared between them and regular twitcher mice. Twitcher/IL-6-deficient mice had a more severe disease than regular twitcher mice: they had an earlier onset day of twitching, a greater number of PAS-positive cells, a greater susceptibility to LPS, an exaggerated gliotic response around some vessels, an elevated level of TNF-alpha, and a compromised blood-brain barrier, which was evaluated by three independent measures. This latter finding indicates that IL-6 plays a role in maintaining the integrity of the BBB, and it raises the possibility that IL-6 functions in a similar manner in other diseases of the CNS. LPS was found to greatly shorten the life of twitcher and twitcher/IL-6-deficient mice compared to genotyped-matched saline-injected mice. This result indicates that a proinflammatory condition can exacerbate an underlying CNS pathology, which could help explain why some leukodystrophy patients display their initial symptoms following a fever or blow to the head.

Peroxisome Proliferator-Activated Receptor β/δ Expression and Activation in Lung Cancer

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is a ligand-binding inducible transcriptional factor linked to carcinogenesis. Important functions of PPARbeta/delta were demonstrated in series of human epithelial cancers; however, its role in lung cancer remains controversial. We investigated the differential expression level and localization of PPARbeta/delta in tumors and adjacent normal lung tissue, and the effect of PPARbeta/delta activation on lung cancer cell proliferation and apoptosis. PPARbeta/delta was expressed in all studied human non-small cell lung cancers, and strong PPARbeta/delta immunoreactivity was observed in epithelial cells of more than 75% of studied lung tumors. PPARbeta/delta expression was consistently limited to the cancer cells in tumor tissue, while in adjacent normal lung tissue it was limited predominantly to the mononuclear cells. We found that ligand-binding activation of PPARbeta/delta stimulates cell proliferation (an effect that was blocked by a dominant-negative construct of PPARbeta/delta), stimulates anchorage-independent cell growth, and inhibits apoptosis in lung cancer cell lines. Importantly, the activation of PPARbeta/delta induces Akt phosphorylation correlated with up-regulation of PDK1, down-regulation of PTEN, and increased expression of Bcl-xL and COX-2. These findings indicate that PPARbeta/delta exerts proliferative and anti-apoptotic effects via PI3K/Akt1 and COX-2 pathways. In conclusion, PPARbeta/delta is strongly expressed in the majority of lung cancers, and its activation induces proliferative and survival response in non-small cell lung cancer.

Neurotrimin is an estrogen-regulated determinant of peripheral sympathetic innervation

Mechanisms underlying axon degeneration in peripheral neuropathies and during normal remodeling are poorly understood. Because estrogen induces widespread sympathetic axon degeneration in female reproductive tract smooth muscle, we surveyed estrogen‐regulated genes in rat myometrium. Microarray analysis revealed that the neural cell adhesion protein neurotrimin (Ntm) was markedly up‐regulated 6 hr and down‐regulated 24 hr after injection of 17β‐estradiol, and real time RT‐PCR confirmed this pattern of expression. Protein analysis by Western blotting showed that uterine Ntm protein is also up‐regulated in vivo 6–24 hr following estrogen injection and that Ntm protein is increased selectively in the myometrium during the high‐estrogen phase of the estrous cycle. Cultured myometrial smooth muscle cells display perinuclear accumulations of Ntm protein, and 17β‐estradiol also increases intracellular levels of Ntm and its secretion into the culture medium. To determine if neurotrimin is required for estrogen‐induced sympathetic pruning, sympathetic neurons were cocultured with uterine smooth muscle cells transfected with siRNA directed against Ntm. Although estrogen inhibited neurite outgrowth in nontransfected cocultures, estrogens ability to reduce sympathetic outgrowth was impaired substantially following Ntm down‐regulation. This supports a role for neurotrimin in mediating estrogen‐induced sympathetic pruning in some peripheral targets. Together with earlier studies, these findings support the idea that physiological sympathetic axon degeneration is a multifactorial process requiring dynamic regulation of multiple repellant proteins.

High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter–driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

Increased salt consumption induces body water conservation and decreases fluid intake

BACKGROUND. The idea that increasing salt intake increases drinking and urine volume is widely accepted. We tested the hypothesis that an increase in salt intake of 6 g/d would change fluid balance in men living under ultra-long-term controlled conditions. METHODS. Over the course of 2 separate space flight simulation studies of 105 and 205 days’ duration, we exposed 10 healthy men to 3 salt intake levels (12, 9, or 6 g/d). All other nutrients were maintained constant. We studied the effect of salt-driven changes in mineralocorticoid and glucocorticoid urinary excretion on day-to-day osmolyte and water balance. RESULTS. A 6-g/d increase in salt intake increased urine osmolyte excretion, but reduced free-water clearance, indicating endogenous free water accrual by urine concentration. The resulting endogenous water surplus reduced fluid intake at the 12-g/d salt intake level. Across all 3 levels of salt intake, half-weekly and weekly rhythmical mineralocorticoid release promoted free water reabsorption via the renal concentration mechanism. Mineralocorticoid-coupled increases in free water reabsorption were counterbalanced by rhythmical glucocorticoid release, with excretion of endogenous osmolyte and water surplus by relative urine dilution. A 6-g/d increase in salt intake decreased the level of rhythmical mineralocorticoid release and elevated rhythmical glucocorticoid release. The projected effect of salt-driven hormone rhythm modulation corresponded well with the measured decrease in water intake and an increase in urine volume with surplus osmolyte excretion. CONCLUSION. Humans regulate osmolyte and water balance by rhythmical mineralocorticoid and glucocorticoid release, endogenous accrual of surplus body water, and precise surplus excretion. FUNDING. Federal Ministry for Economics and Technology/DLR; the Interdisciplinary Centre for Clinical Research; the NIH; the American Heart Association (AHA); the Renal Research Institute; and the TOYOBO Biotechnology Foundation. Food products were donated by APETITO, Coppenrath und Wiese, ENERVIT, HIPP, Katadyn, Kellogg, Molda, and Unilever.

Lipopolysaccharide-dependent interaction between PU.1 and cJun determines production of lipocalin-type prostaglandin D synthase and prostaglandin D2 in macrophages

Previously, we reported that expression of lipocalin-prostaglandin D synthase (L-PGDS) is inducible in macrophages and protects from Pseudomonas pneumonia. Here, we investigated the mechanism by which L-PGDS gene expression is induced in macrophages. A promoter analysis of the murine L-PGDS promoter located a binding site of PU.1, a transcription factor essential for macrophage development and inflammatory gene expression. A chromatin immunoprecipitation assay showed that PU.1 bound to the cognate site in the endogenous L-PGDS promoter in response to LPS. Overexpression of PU.1, but not of PU.1(S148A), a mutant inert to casein kinase II (CKII) or NF-kappaB-inducing kinase (NIK), induced L-PGDS in RAW 264.7 cells. Conversely, siRNA silencing of PU.1 expression blunted productions of L-PGDS and prostaglandin D2 (PGD(2)). LPS treatment induced formation of the complex of PU.1 and cJun on the PU.1 site, but inactivation of cJun by treatment with JNK or p38 kinase inhibitor abolished the complex, and suppressed PU.1 transcriptional activity for L-PGDS gene expression. Together, these results show that PU.1, activated by CKII or NIK, cooperates with MAPK-activated cJun to maximally induce L-PGDS expression in macrophages following LPS treatment, and suggest that PU.1 participates in innate immunity through the production of L-PGDS and PGD(2).

Apoferritin attenuates experimental allergic encephalomyelitis in SJL mice

Ferritin has been shown to attenuate iron-catalyzed oxidative damage in several experimental conditions. Since oxidative damage has been implicated in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), an animal model of MS, we tested the hypothesis that ferritin would act to attenuate disease. The experimental design was to increase plasma ferritin levels during the active stage of EAE by giving systemic injections of apoferritin and then compare disease activity between these mice and EAE mice administered vehicle. Additional mice received systemic injections of iron, which induces ferritin synthesis, in order to test the effects of exogenous iron on the disease course. Although plasma levels of ferritin were found to be elevated in both apoferritin and iron-injected EAE mice, only apoferritin treatment resulted in a reduction in disease activity compared to EAE mice given vehicle. The suppressive effects of apoferritin administration suggest that the increase in endogenous ferritin levels that have been previously observed in the cerebrospinal fluid of chronic progressive MS patients with active disease might be functioning to limit the severity and spread of tissue damage.