Extensive elimination of acinar cells during normal postnatal pancreas growth.

While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture.

Unique features of the arterial blood-brain barrier.

CNS vasculature differs from vascular networks of peripheral organs by its ability to tightly control selective material exchange across capillary barriers. Capillary permeability is mostly defined by unique cellular components of the endothelium. While capillaries are extensively investigated, the barrier properties of larger vessels are understudied. Here, we investigate barrier properties of CNS arterial walls. Using tracer challenges and various imaging modalities, we discovered that at the mouse cortex, the arterial barrier does not reside at the classical level of the endothelium. The arterial wall's unique permeability acts bi-directionally; CSF substances travel along the glymphatic path and can penetrate from the peri-vascular space through arteriolar walls towards the lumen. We found that caveolae vesicles in arteriole endothelial are functional transcytosis machinery components, and that a similar mechanism is evident in the human brain. Our discoveries highlight vascular heterogeneity investigations as a potent approach to uncover new barrier mechanisms.

P450 oxidoreductase regulates barrier maturation by mediating retinoic acid metabolism in a model of the human BBB.

The blood-brain barrier (BBB) selectively regulates the entry of molecules into the central nervous system (CNS). A crosstalk between brain microvascular endothelial cells (BMECs) and resident CNS cells promotes the acquisition of functional tight junctions (TJs). Retinoic acid (RA), a key signaling molecule during embryonic development, is used to enhance in vitro BBB models' functional barrier properties. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) regulates the enzymatic activity of microsomal cytochromes. POR-deficient (PORD) patients display impaired steroid homeostasis and cognitive disabilities. Here, we used both patient-specific POR-deficient and CRISPR-Cas9-mediated POR-depleted induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) to study the role of POR in the acquisition of functional barrier properties. We demonstrate that POR regulates cellular RA homeostasis and that POR deficiency leads to the accumulation of RA within iBMECs, resulting in the impaired acquisition of TJs and, consequently, to dysfunctional development of barrier properties.

Nano-scale architecture of blood-brain barrier tight-junctions.

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.

Angiomodulin (IGFBP7) is a cerebral specific angiocrine factor, but is probably not a blood-brain barrier inducer.

BACKGROUND: Several secreted factors have been identified as drivers of cerebral vasculature development and inducers of blood-brain barrier (BBB) differentiation. Vascular endothelial growth factor A (VEGF-A) is central for driving cerebral angiogenesis and Wnt family factors (Wnt7a, Wnt7b and norrin) are central for induction and maintenance of barrier properties. Expressed by developing neural tissue (neuron and glia progenitors), they influence the formation of central nervous system (CNS) vascular networks. Another type of factors are tissue-specific paracrine factors produced by endothelial cells (ECs), also known as 'angiocrine' factors, that provide instructive signals to regulate homeostatic and regenerative processes. Very little is known about CNS angiocrine factors and their role in BBB development. Angiomodulin (AGM) was reported to be expressed by developing vasculature and by pathological tumor vasculature. Here we investigated AGM in the developing CNS and its function as a potential BBB inducer. METHODS: We analyzed microarray data to identify potential angiocrine factors specifically expressed at early stages of barrier formation. We then tested AGM expression with immunofluorescence and real-time PCR in various organs during development, post-natal and in adults. Permeability induction with recombinant proteins (Miles assay) was used to test potential interaction of AGM with VEGF-A. RESULTS: Several angiocrine factors are differentially expressed by CNS ECs and AGM is a prominent CNS-specific angiocrine candidate. Contrary to previous reports, we found that AGM protein expression is specific to developing CNS endothelium and not to highly angiogenic developing vasculature in general. In skin vasculature we found that AGM antagonizes VEGF-A-induced vascular hyperpermeability. Finally, CNS AGM expression is not specific to BBB vasculature and AGM is highly expressed in non-BBB choroid-plexus vasculature. CONCLUSIONS: We propose AGM as a developmental CNS vascular-specific marker. AGM is not a pan-endothelial marker, nor a general marker for developing angiogenic vasculature. Thus, AGM induction in the developing CNS might be distinct from its induction in pathology. While AGM is able to antagonize VEGF-A-induced vascular hyperpermeability in the skin, its high expression levels in non-BBB CNS vasculature does not support its potential role as a BBB inducer. Further investigation including loss-of-function approaches might elucidate AGM function in the developing CNS.

Leptin receptor deficiency induces early, transient and hyperglycaemia-independent blood-brain barrier dysfunction.

Diabetes mellitus (DM) significantly increases susceptibility to central nervous system (CNS) pathologies, including stroke, vascular dementia, cognitive deficits and Alzheimer's disease. Previous studies (mostly using the streptozotocin model) suggested that blood-brain barrier (BBB) disruption is involved in these conditions. Here, we examined the integrity of brain capillaries and BBB permeability in Leprdb/db obesity-related diabetic mice. Surprisingly, significant BBB leakage was observed only in young mice at the pre-hyperglycemic stage. Thorough examination of barrier permeability at later diabetic stages showed no evidence for significant BBB leakage during the hyperglycemic state. Electron microscopy imaging of mice with short-term hyperglycaemia supported normal BBB permeability but indicated other stress-related changes in capillary ultrastructure, such as mitochondrial degeneration. Based on our study with this mouse genetic model of obesity-related DM, we suggest that previously reported hyperglycaemia-induced BBB leakage is most likely not the underlying mechanism of DM-related CNS pathologies. Finally we propose that BBB hyper-permeability might be an early and transient phenomenon while stress-related endothelial pathologies do correlate with a short-term diabetic state.

Postnatal Exocrine Pancreas Growth by Cellular Hypertrophy Correlates with a Shorter Lifespan in Mammals.

Developmental processes in different mammals are thought to share fundamental cellular mechanisms. We report a dramatic increase in cell size during postnatal pancreas development in rodents, accounting for much of the increase in organ size after birth. Hypertrophy of pancreatic acinar cells involves both higher ploidy and increased biosynthesis per genome copy; is maximal adjacent to islets, suggesting endocrine to exocrine communication; and is partly driven by weaning-related processes. In contrast to the situation in rodents, pancreas cell size in humans remains stable postnatally, indicating organ growth by pure hyperplasia. Pancreatic acinar cell volume varies 9-fold among 24 mammalian species analyzed, and shows a striking inverse correlation with organismal lifespan. We hypothesize that cellular hypertrophy is a strategy for rapid postnatal tissue growth, entailing life-long detrimental effects.

Mechanisms of neuropsychiatric lupus: The relative roles of the blood-cerebrospinal fluid barrier versus blood-brain barrier.

The pathogenesis of neuropsychiatric lupus (NPSLE) is believed to include the entry of circulating neuropathic antibodies to the brain via a pathologically permeable blood-brain barrier (BBB). Nevertheless, direct evidence of BBB pathology or mechanisms underlying BBB dysfunction is missing. Here, we examined BBB integrity in an established NPSLE mouse model (MRL/faslpr/lpr). Surprisingly, challenging the barrier with various exogenous tracers demonstrated insignificant changes in BBB permeability. Furthermore, electron microscopy showed no ultrastructure changes supporting hyper-permeability. However, we found that abnormal function of the blood-cerebrospinal fluid barrier (BCSFB) in the choroid plexus underlies brain exposure to neuropathic antibodies. Considerable intrathecal lymphocyte infiltration likely occurs through the BCSFB, accompanied by epithelial hyper-permeability to antibodies. Our results challenge the commonly held view of BBB disruption in NPSLE, supporting a shift in focus to BCSFB dysfunction as a causative factor in the disease.

p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Can nitroxides evoke the Keap1-Nrf2-ARE pathway in skin?

Nitroxides are stable cyclic radicals of diverse size, charge, and lipophilicity. They are cell-permeative, which effectively protects cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The mechanisms of activity through which nitroxides operate are diverse, including superoxide dismutase-mimetic activity, oxidation of semiquinone radicals, oxidation of reduced metal ions, procatalase-mimetic activity, interruption of radical chain reactions, and indirect modulation of NO levels. Nitroxides possess both a nucleophilic (reducing properties) and an electrophilic (oxidizing properties) nature and, therefore, they may affect different cellular pathways. In the current study, a novel mechanism of action by which nitroxides provide skin protection based on their electrophilic nature is suggested. This study shows that nitroxides may act as electrophiles, directly or indirectly, capable of activating the Keap1-Nrf2-ARE pathway in human keratinocytes (HaCaT) and in human skin (human organ culture model). The high potency of oxoammonium cations versus hydroxylamines in activating the system is demonstrated. The mechanism of action by which nitroxides activate the Keap1-Nrf2-ARE pathway is discussed. Understanding the mechanism of activity may expand the usage of nitroxides as a skin protection strategy against oxidative stress-related conditions.

MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation.

Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the DeltaNp63alpha protein. We found that MDM2 binds DeltaNp63alpha in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for DeltaNp63alpha nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of DeltaNp63alpha by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the alpha and beta tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous DeltaNp63alpha in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative DeltaNp63alpha protein.

Jun proteins are starvation-regulated inhibitors of autophagy.

The growing number of biological functions affected by autophagy ascribes a special significance to identification of factors regulating it. The activator protein-1 (AP-1) transcription factors are involved in most aspects of cellular proliferation, death, or survival, yet no information regarding their involvement in autophagy is available. Here, we show that the AP-1 proteins JunB and c-Jun, but not JunD, c-Fos, or Fra-1, inhibit autophagy. JunB inhibits autophagy induced by starvation, overexpression of a short form of ARF (smARF), a potent inducer of autophagy, or even after rapamycin treatment. In agreement, acute repression of JunB expression, by JunB knockdown, potently induces autophagy. As expected from autophagy-inhibiting proteins, Jun B and c-Jun expression is reduced by starvation. Decrease in JunB mRNA expression and posttranscriptional events downregulate JunB protein expression after starvation. The inhibition of autophagy by JunB is not mediated by mammalian target of rapamycin (mTOR) regulation, as it occurs also in the absence of mTOR activity, and autophagy induced by JunB knockdown is not correlated with changes in mTOR activity. Nevertheless, the transcriptional activities of c-Jun and JunB are required for autophagy inhibition, and JunB incapable of heterodimerizing is a less effective inhibitor of autophagy. Most importantly, inhibition of autophagy in starved HeLa cells by JunB enhances apoptotic cell death. We suggest that JunB and c-Jun are regulators of autophagy whose expression responds to autophagy-inducing signals.

DNA damage-dependent translocation of B23 and p19 ARF is regulated by the Jun N-terminal kinase pathway.

The dynamic behavior of the nucleolus plays a role in the detection of and response to DNA damage of cells. Two nucleolar proteins, p14(ARF)/p19(ARF) and B23, were shown to translocate out of the nucleolus after exposure of cells to DNA-damaging agents. This translocation affects multiple cellular functions, such as DNA repair, proliferation, and survival. In this study, we identify a pathway and scrutinize the mechanisms leading to the translocation of these proteins after exposure of cells to DNA-damaging agents. We show that redistribution of B23 and p19(ARF) after the exposure to genotoxic stress occurs preferentially when the c-Jun-NH(2)-kinase (JNK) pathway is activated and is inhibited when the JNK pathway is impaired. The stress-induced translocation of alternative reading frame (ARF) is JNK dependent and mediated by two activator proteins, c-Jun and JunB. Thr(91) and Thr(93) of c-Jun are required for the translocation, but the transcriptional activity of c-Jun is dispensable. Instead, c-Jun interacts with B23 in a dose-dependent manner. c-Jun itself is excluded from the nucleolus in a JNK-dependent manner. Hence, we suggest that c-Jun translocates B23 and ARF from the nucleolus after JNK activation by means of protein interactions. In senescent cells, JNK activity and c-Jun levels are reduced concomitantly with ARF nucleolar accumulation, and UV radiation does not cause the translocation of ARF.

Transcriptional repression of c-Jun's E3 ubiquitin ligases contributes to c-Jun induction by UV.

UV radiation is a major environmental carcinogen. The oncoprotein c-Jun that is required for development of skin cancer is stabilized by UV radiation. The mechanism leading to its stabilization after exposure to UV is not known. The lack of knowledge was particularly sharpened, after the discovery that JNK, the most potent positive regulator of c-Jun, activates Itch, an E3-ligase of c-Jun and JunB. In this study we demonstrate that the expression of all three E3 ubiquitin ligases of c-Jun is down-regulated by UV. The levels of Itch/AIP4 and Fbw7alpha transcripts are reduced following UV exposure in every cell line examined. Repression of hCOP1 and its associated protein hDET1, which is required for c-Jun degradation, is cell type dependent. Expression of Fbw7alpha is down-regulated by UVC or UVB, independently of the p53, MAPK and the PKC pathways but the repression is inhibited in the absence of active Fbw7 proteins suggesting that a target protein of Fbw7 is involved in Fbw7 expression/repression. The repression does not require protein synthesis and UV does not change Fbw7 mRNA stability. The characteristics of Fbw7alpha repression perfectly match with those of c-Jun induction. Unlike UV, ionizing radiation does not repress Fbw7alpha and does not induce c-Jun. In addition, the repression kinetics correlates tightly with the kinetics of c-Jun induction by UV. Moreover, abrogation of Fbw7 UV-responsiveness abolishes c-Jun induction by UV, and knockdown of Fbw7 results in elevated basal expression of c-Jun but reduced UV-dependent induction thus, proving the essential role of this repression in c-Jun induction by UV.

Induction of transcriptionally active Jun proteins regulates drug-induced senescence.

The drug hydroxyurea (HU) is used for cancer therapy and treatment of sickle cell anemia. It inhibits cell cycle progression by blocking DNA synthesis and drives cells to undergo apoptosis or enter senescence. We demonstrate here that HU induces the expression of two AP-1 proteins, c-Jun and JunB, which exert antagonistic effects on the cell cycle. Moreover, the induction of c-Jun is observed following treatment with two other drugs that inhibit the cell cycle in S phase, aphidicolin and camptothecin. The induction of c-Jun, which promotes cell cycle progression, up-regulates expression of cyclin D after exposure of cells to HU. Deficiency in c-jun prevents elevation of cyclin D expression and extends entrance into HU-induced senescence but also renders cells more resistant to HU-dependent apoptosis. The induction of c-Jun is independent of JNK activity, and additionally, of c-Jun autoregulatory activity but is inhibited upon inhibition of protein kinase C activity. Therefore, we suggest that c-Jun activity prevents drug-induced senescence. Conversely, the JunB target gene, tumor suppressor p16(INK4a), a cyclin-dependent kinase inhibitor essential for the induction of drug-induced senescence, is also up-regulated by HU in a JunB-dependent manner. Constitutive expression of JunB up-regulates p16(INK4a) and increases the sensitivity of mouse fibroblasts to drug-induced-senescence. Thus, we suggest that in contrast to c-Jun, JunB drives cells to enter HU-dependent senescence. The effect of HU treatment, which regulates the intricate web of AP-1 transcription, depends on the balance between c-Jun and JunB activities.