Necdin controls proliferation of white adipocyte progenitor cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Rem2 GTPase controls proliferation and apoptosis of neurons during embryo development

We have recently found that Rem2 GTPase, highly expressed in human embryonic stem cells (hESC), maintains the cell cycle and controls proper differentiation towards ectoderm, suggesting a role in neuronal development. We describe here the use of the zebrafish (Danio Rerio) model to determine the physiological significance of Rem2 during embryogenesis. We show that Rem2 RNA is highly expressed in zebrafish embryos up to 2 hours of development followed by a decrease in expression until 48 hours when afterwards Rem2 is switched on again until 5 days. In situexpression analysis reveals that Rem2 is expressed exclusively in the tectum of the brain and eye of the zebrafish. Rem2 morpholino demonstrates impaired embryo development resulting in loss of neural tissue.     

The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

Regulating proliferation during retinal development

Recent studies have shown that components of the cell-cycle machinery can have diverse and unexpected roles in the retina. Cyclin-kinase inhibitors, for example, have been implicated as regulators of cell-fate decisions during histogenesis and reactive gliosis in the adult tissue after injury. Also, various mechanisms have been identified that can compensate for extra rounds of cell division when the normal timing of the cell-cycle exit is perturbed. Surprisingly, distinct components of the cell-cycle machinery seem to be used during different stages of development, and different organisms might rely on distinct pathways. Such detailed studies on the regulation of proliferation in complex multicellular tissues during development have not only advanced our knowledge of the ways in which proliferation is controlled, but might also help us to understand the degenerative disorders that are associated with gliosis and some types of tumorigenesis.     

The COP9 signalosome regulates Skp2 levels and proliferation of human cells

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCF(Skp2), and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27(Kip1). CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27(Kip1). Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCF(Skp2) complex and therefore contributes to cell cycle regulation of human cells.     

Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation

The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy.     

Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation

We have identified a new gene, Zfrp8, as being essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of gamma-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring gamma-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, we propose that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation.     

2-methoxyestradiol,an endogenous metabolite of 17b-estradiol,inhibits adipocyte proliferation

The effects of 2-methoxyestradiol (2ME), a naturally occurring mammalian metabolite of 17 -estradiol, on adipocyte growth has been investigated in mouse brown adipose tissue precursor cells developed in primary culture. 2ME inhibits brown adipocyte proliferation in a dose-response manner (IC50 = 1.7 × 10-6 M for DNA synthesis), with much higher potency than its hormone precursor 17-estradiol, and cells acquire the typical differentiated morphology - more round with a higher content of triglycerides. 2ME causes similar effects in the immortal brown adipocyte tumor-derived hibernoma cell line HIB 1B and the immortal 3T3-F442A white adipocyte line. These findings suggest a possible role for 2ME in adipocyte proliferation, and probably in the differentiation process, entering the cells in the adipogenic program.     

Control of cell proliferation during plant development

Knowledge of the control of cell division in eukaryotes has increased tremendously in recent years. The isolation and characterization of the major players from a number of systems and the study of their interactions have led to a comprehensive understanding of how the different components of the cell cycle apparatus are brought together and assembled in a fine-tuned machinery. Many parts of this machine are highly conserved in organisms as evolutionary distant as yeast and animals. Some key regulators of cell division have also been identified in higher plants and have been shown to be functional homologues of the yeast or animal proteins. Although still in its early days, investigations into the regulation of these molecules have provided some clues on how cell division is coupled to plant development.     

Cell proliferation patterns during development of the equine placenta

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.     

The paracaspase MALT1 controls caspase-8 activation during lymphocyte proliferation

Caspase-8, an initiator caspase involved in lymphocyte apoptosis, is paradoxically required for lymphocyte proliferation. It is not understood how caspase-8 is controlled during antigenic signaling to allow for activation while averting the triggering of apoptosis. Here, we show that caspase-8 undergoes limited activation upon antigenic stimulation, and this activation is dependent on the paracaspase MALT1. The paracaspase domain of MALT1, in a protease-independent manner, induces caspase-8 activation through direct association. MALT1 diminishes the activation of apoptotic effector caspases, but it does not alter the activity of caspase-8 toward c-FLIP(L), which is required for antigenic signaling. Mutants of MALT1 that fail to activate caspase-8 and permit c-FLIP(L) cleavage cannot facilitate NF-kappaB activation or IL-2 induction. Our results reveal a mechanism that utilizes a protease potentially deadly to the cell for proliferative signaling and demonstrate a functional connection between the caspase and paracaspase families to enable nonapoptotic processes.