MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS's functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCß KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCß KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.

Activation of GSK3 Prevents Termination of TNF-Induced Signaling.

BACKGROUND: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context. MATERIALS AND METHODS: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/ß-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays. RESULTS: Constitutive GSK3ß and PKCß expression and GSK3α/ß-Ser21/9 and PKCα/ßII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/ß-knockdown modestly increased, whereas GSK3α/ß-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/ß-Ser176/180 phosphorylation were not affected. Moreover, PKCß-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects. CONCLUSION: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 - either pathophysiologically or pharmacologically induced - may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.

GSK3: A Kinase Balancing Promotion and Resolution of Inflammation.

GSK3 has been implicated for years in the regulation of inflammation and addressed in a plethora of scientific reports using a variety of experimental (disease) models and approaches. However, the specific role of GSK3 in the inflammatory process is still not fully understood and controversially discussed. Following a detailed overview of structure, function, and various regulatory levels, this review focusses on the immunoregulatory functions of GSK3, including the current knowledge obtained from animal models. Its impact on pro-inflammatory cytokine/chemokine profiles, bacterial/viral infections, and the modulation of associated pro-inflammatory transcriptional and signaling pathways is discussed. Moreover, GSK3 contributes to the resolution of inflammation on multiple levels, e.g., via the regulation of pro-resolving mediators, the clearance of apoptotic immune cells, and tissue repair processes. The influence of GSK3 on the development of different forms of stimulation tolerance is also addressed. Collectively, the role of GSK3 as a kinase balancing the initiation/perpetuation and the amelioration/resolution of inflammation is highlighted.

Postnatal growth curves for extremely low birth weight infants with early enteral nutrition.

UNLABELLED: Early enteral nutrition improves growth of extremely low birth weight infants, but growth curves beyond 30 days of life are lacking for such infants receiving early enteral nutrition. Based on the data of all infants born in a 4-year interval with a birth weight <1000 g and surviving for >56 days, we calculated growth rates and weight gain over 120 postnatal days. Infants with major congenital anomalies or necrotising enterocolitis were excluded. Daily weight, weekly length, head circumference and nutritional data were collected until discharge or for maximal 120 days. Curves were calculated in 100 g birth weight intervals, and separately for appropriate for gestational age (AGA) and small for gestational age (SGA) infants. Data were available from 163 infants (birth weight 768 g +/- 153 g; gestational age 26.8+/-1.8 weeks; mean +/- SD) including 55 SGA infants (33.7%). Full enteral feeding was achieved at day 21.7 (+/-10.4). After 12.8% (+/-6.6%) maximal postnatal weight loss at day 7.5 (+/-3.0), birth weight was regained at 14.6 (+/-6.0) days. Mean overall weight gain was 15 g/kg per day with a significantly higher weight gain for SGA than for AGA infants (P <0.05). CONCLUSION: Our early fed infants achieved better weight gain than those recently published receiving late enteral nutrition, but nevertheless fell below the 10th percentile of intrauterine curves. Which postnatal growth is ideal for extremely low birth weight infants infants is unclear. Our growth curves should not be taken as reference curves of a "normal population" but may help to identify infants with growth failure.