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The CsdA protein is a highly conserved, DEAD-box RNA helicase and assists RNA structural remodeling at low temperature. We show that the fast-growing wild-type (WT) cells contain higher number of replication origins per cell with bigger cell size and the slowly growing cells possess less number of replication origins per cell with smaller cell size. The absence of CsdA leads to production of larger cells with higher number of origins per cell but slower growth at low temperature in an independent-manner of growth media. The phenotypes in ΔcsdA mutant are reversed by ectopic expression of CsdA or RNase R. A global transcription analysis shows that the absence of CsdA leads to significant decreases in transcription of about 200 genes at low temperature. These genes are associated with essential metabolic pathways, flagger assembly and cell division (minDE). It is likely that the slow growth of ΔcsdA cell results from the decreased transcription of essential metabolic genes, and the larger ΔcsdA cell could be a result of decreased transcription of minDE. The increased transcription of the nrdHIEF genes in ΔcsdA mutant is a likely reason that promotes DNA replication. We conclude that CsdA coordinates the cell cycle to growth by stabilizing mRNA of essential metabolic and cell division genes and degrading mRNA for nucleotide metabolic genes at low temperature.

In most eukaryotes, size homeostasis is exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. At the cellular level, size is dictated by the balance between cellular growth and division such that each cell division is accompanied by a doubling in cell mass. Our systematic screen for size mutants revealed that hundreds of genes markedly altered cell size in the opportunistic yeast Candida albicans, but only few of these overlapped with size control genes in the model yeast Saccharomyces cerevisiae. Here, we characterized one of the potent size regulators in C. albicans, the zinc-finger transcription factor Ahr1 that is unique to Candida yeasts of the CTG-clade. We found that Ahr1 acts as both a repressor of Start and a transcriptional regulator of amino acid metabolic genes. Consistently, Ahr1 was required for amino acid and nitrogen-source modulation of cell size. Genetic interactions with deletions of different known Start regulators in C. albicans revealed functional relationship of Ahr1 with the AGC family protein kinase Sch9. Collectively, this work uncovered a novel network of the nutrient-dependent size control in C. albicans and emphasizes the impact of nitrogen and amino acid metabolisms in size homeostasis in this pathogenic fungus.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC1 and PC2 localize to the primary cilium and form a protein complex, which is thought to regulate signaling events. PKD1 mutations are associated with a stronger phenotype than PKD2, suggesting the existence of PC1 specific functions in renal tubular cells. However, the evidence for diverging molecular functions is scant. The bending of cilia by fluid flow induces a reduction in cell size through a mechanism that involves the kinase LKB1 but not PC2. Here, using different in vitro approaches, we show that contrary to PC2, PC1 regulates cell size under flow and thus phenocopies the loss of cilia. PC1 is required to couple mechanical deflection of cilia to mTOR in tubular cells. This study pinpoints divergent functions of the polycystins in renal tubular cells that may be relevant to disease severity in ADPKD.

Granulocyte-colony stimulating factor (G-CSF) is a cytokine crucially involved in the regulation of granulopoiesis and the mobilization of hematopoietic stem cells from bone marrow. However, emerging data suggest that G-CSF exhibits more diverse functions than initially expected, such as conferring protection against apoptosis to neural cells and stimulating mitogenesis in cardiomyocytes and skeletal muscle stem cells after injury. In the present study, we sought to investigate the potential contribution of G-CSF to the regulation of muscle volume. We found that the administration of G-CSF significantly enhances muscle hypertrophy in two different muscle overload models. Interestingly, there was a significant increase in the transcripts of both G-CSF and G-CSF receptors in the muscles that were under overload stress. Using mutant mice lacking the G-CSF receptor, we confirmed that the anabolic effect is dependent on the G-CSF receptor signaling. Furthermore, we found that G-CSF increases the diameter of myotubes in vitro and induces the phosphorylation of AKT, mTOR, and ERK1/2 in the myoblast-like cell line C2C12 after differentiation induction. These findings indicate that G-CSF is involved in load-induced muscle hypertrophy and suggest that G-CSF is a potential agent for treating patients with muscle loss and sarcopenia.

Oral squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a high incidence of recurrence and distant metastasis. However, the mechanism of epithelial to mesenchymal transition (EMT) during tumor progression and metastasis in OSCC has not yet been fully elucidated. It is well known that the Cl

Liraglutide (LRG), a glucagon-like peptide 1 analogue (GLP1A), could decrease body mass of type 2 diabetes (T2DM), but the exact molecular mechanism of LRG has not been elucidated. This study was performed to explore whether LRG regulated TG synthesis via secretion of FGF21 and modulating AMP-dependent protein kinase (AMPK) pathway in an autocrine mode. Two-month-old male C57BL/6 mice were fed high-fat diet (HFD) for 4 months followed by injection of 30 mg/kg streptozotocin (STZ) to induce state of T2DM. Then DM mice were given LRG (0.4 mg/kg/d) for 4 months. Body mass, serum lipids and FGF21 levels, related gene expression were analyzed. Lipopolysaccharide (LPS)-induced RAW264.7 cells were treated with palmitic acid and different concentrations of LRG. Then Exendin (9–39), siRNA targeted to liver kinase B1 (LKB1) and Compound C were used to confirm the signaling pathway. LRG decreased adipocyte size, increased secretion of FGF21, and promoted phosphorylation of LKB1, AMPK and Acetyl coenzyme A carboxylase 1 (ACC1) in white adipose tissue (WAT) of DM mice. LRG also increased phosphorylation of fibroblast growth factor receptor 3 (FGFR3), LKB1, AMPK and ACC1 via FGF21 secretion, which ultimately inhibited synthesis of TG in macrophage. In conclusion, FGF21 is induced to be expressed in macrophage by LRG, which then activates LKB1-AMPK-ACC1 pathway in an autocrine manner.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl

There is increasing evidence that the sympathetic nervous system (SNS) plays an important role in adipose tissue development. However, the underlying molecular mechanism(s) associated with this remains unclear. SNS innervation of white adipose tissue (WAT) is believed to be necessary and sufficient to elicit WAT lipolysis. In this current study, mice with Schwann cell (SC)-specific inactivation of phosphatase and tensin homolog (Pten) displayed enlarged inguinal white adipose tissue (iWAT). This serendipitous observation implicates the role of SCs in mediating SNS activity associated with mouse adipose tissue development. Mice with SC-specific Pten inactivation displayed enlarged iWAT. Interestingly, the SNS activity in iWAT of SC-specific Pten-deficient mice was reduced as demonstrated by decreased tyrosine hydroxylase (TH) expression level and neurotransmitters, such as norepinephrine (NE) and histamine (H). The lipolysis related protein, phosphorylated hormone sensitive lipase (pHSL), was also decreased. As expected, AKT-associated signaling pathway was hyperactivated and hypothesized to induce enlarged iWAT in SC-specific Pten-deficient mice. Moreover, preliminary experiments using AKT inhibitor AZD5363 treatment ameliorated the enlarged iWAT condition in SC-specific Pten-deficient mice. Taken together, SCs play an essential role in the regulation of SNS activity in iWAT development via the AKT signaling pathway. This novel role of SCs in SNS function allows for better understanding into the genetic mechanisms of peripheral neuropathy associated obesity.

The extracellular matrix (ECM) stiffness has direct effect on cancer cells homeostasis (e.g., cell volume), which is critical for regulation of their migration. However, the relationship among ECM stiffness, cell volume and cancer cell migration in three-dimensional (3D) microenvironment remains elusive. In this work, we prepared the collagen-alginate hydrogels with tunable stiffness to study how the 3D ECM stiffness influences cell volume and their migration. We found the cell volume homeostasis and migration speed of the MDA-MB-231 cells are both regulated by 3D ECM stiffness, while cell migration speed shows the same stiffness-dependent trend with cell volume. Deviating the cell volume from its homeostasis state can cause a significant decrease in its migration ability, which can be recovered through recovering the cell volume to its homeostasis state. This work reveals for the first time that 3D ECM stiffness regulates cell migration behavior through regulating cell volume homeostasis, which may provide a novel view in the exploration of the underlying mechanisms of cancer metastasis and cellular mechanotransduction.

Bone mass is determined by coordinated acts of osteoblasts and osteoclasts, which control bone formation and resorption, respectively. Osteoclasts are multinucleated, macrophage/monocyte lineage cells from bone marrow. The Dok-family adaptors Dok-1, Dok-2 and Dok-3 are expressed in the macrophage/monocyte lineage and negatively regulate many signaling pathways, implying roles in osteoclastogenesis. Indeed, mice lacking Dok-1 and Dok-2, the closest homologues with redundant functions, develop osteopenia with increased osteoclast counts compared to the wild-type controls. Here, we demonstrate that Dok-3 knockout (KO) mice also develop osteopenia. However, Dok-3 KO, but not Dok-1/-2 double-KO (DKO), mice develop larger osteoclasts within the normal cell-count range, suggesting a distinctive role for Dok-3. Indeed, Dok-3 KO, but not Dok-1/-2 DKO, bone marrow-derived cells (BMDCs) generated larger osteoclasts with more nuclei due to augmented cell-to-cell fusion in vitro. In addition, while Dok-1/-2 DKO BMDCs generated more osteoclasts, Dok-1/-2/-3 triple-KO (TKO) BMDCs generated osteoclasts increased in both number and size. Furthermore, Dok-1/-2/-3 TKO mice showed the combined effects of Dok-3 and Dok-1/-2 deficiency: severe osteopenia with more and larger osteoclasts. Together, our findings demonstrate that Dok-3 and Dok-1/-2 play distinctive but cooperative roles in osteoclastogenesis and protect mice from osteopenia, providing physiological and pathophysiological insight into bone homeostasis.

Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. Here we performed a gain-of-function screen and identified larp4B whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. We generated transgenic flies overexpressing wild-type Larp4B or a deletion variant lacking the LA and RRM domains, and demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. We found that the larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. Our results suggest that Larp4B is a negative regulator of dMyc.

Npr1 gene (coding for NPR-A) and Npr2 gene (coding for NPR-B) are identified as intrinsic anti-hypertrophic genes that opposes abnormal cardiac remodeling. However, the functional role of Npr1 and Npr2 genes during cardiac hypertrophic growth is not well understood. Hence, the present investigation was aimed to study the effect of Npr1 and Npr2 gene silencing, respectively on β-AR activation induced cardiac hypertrophic growth in H9c2 cells in vitro . The control, Npr1, and Npr2 gene suppressed H9c2 cells, respectively were treated with ISO (10 −5 M) for 48 h. The mRNA and protein expression profile of NPR-A, NPR-B, PKG-I and cGMP were analyzed by qPCR, Western blotting, ELISA, and immunofluorescence methods, respectively. A marked increase in cell size (30.10 ± 0.51 μm vs 61.83 ± 0.43 μm, 2-fold) accompanied by elevated hypertrophic marker genes ( α-sk and β-MHC 3-fold, respectively) expression was observed in Npr1 gene suppressed H9c2 cells as compared with control cells. In contrast, the Npr2 gene suppression in H9c2 cells neither altered the cell size nor the level of hypertrophic marker genes expression. Upon exposure to Isoproterenol, the Npr1 suppressed H9c2 cells exhibited further increase in cell size (1.5 fold), whereas, no significant increase in cell size or marker genes expression was noticed in Npr2 suppressed cells. Moreover, the intracellular cGMP level was down-regulated by 2-fold in Npr1 suppressed cells, while, no significant change was observed in Npr2 suppressed cells. Together, these results suggest that Npr1 , not Npr2 gene function is positively associated with the initiation of cardiac fetal gene program and development of cardiac hypertrophic growth.

Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria or their descendants, plastids at species-dependent levels. We investigated the physiological significance of the intrinsic SQDG content in the cyanobacterium Synechococcus elongatus PCC 7942, with the use of its mutant, in which the genes for SQDG synthesis, sqdB and sqdX, were overexpressed. The mutant showed a 1.3-fold higher content of SQDG (23.6 mol% relative to total cellular lipids, cf., 17.1 mol% in the control strain) with much less remarkable effects on the other lipid classes. Simultaneously observed were 1.6- to 1.9-fold enhanced mRNA levels for the genes responsible for the synthesis of the lipids other than SQDG, as if to compensate for the SQDG overproduction. Meanwhile, the mutant showed no injury to cell growth, however, cell length was increased (6.1 ± 2.3, cf., 3.8 ± 0.8 μm in the control strain). Accordingly with this, a wide range of genes responsible for cell division were 1.6–2.4-fold more highly expressed in the mutant. These results suggested that a regulatory mechanism for lipid homeostasis functions in the mutant, and that SQDG has to be kept from surpassing the intrinsic content in S. elongatus for repression of the abnormal expression of cell division-related genes and, inevitably, for normal cell division.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Objective —microRNAs (miRNAs) have emerged as novel regulators for cardiac hypertrophy. MiR-122 is well recognized as a promising therapeutic target in liver disease, whereas recently plays important roles in cardiovascular diseases. The current study aimed to explore the effect of miR-122 on the pathogenesis of cardiomyocyte hypertrophy. Methods and results —The cardiomyocytes isolated from the neonatal rat ventricular cardiomyocytes (NRVMs) were collected and performed to Angiotensin II (Ang II) administration. We observed a dramatically increased miR-122 expression in hypertrophic cardiomyocytes. The NRVMs transfected with miR-122 mimic or negative control were utilized for the functional analysis. Overexpression of miR-122 increased the morphology size of cardiomyocytes and promoted the pro-hypertrophic genes expression, whereas downregulated the anti-hypertrophic genes upon Ang II stimulation. The bioinformatics analysis and luciferase reporter assays exhibited that miR-122 directly targeted FoxO3 and attenuated its gene level in hypertrophic cardiomyocytes. Moreover, miR-122 negatively regulated FoxO3 but promoted calcineurin signaling pathway activation. Importantly, FoxO3 overexpression significantly reversed the effect of miR-122 on cardiomyocyte hypertrophy. Conclusion —Collected, our finding demonstrated that miR-122 accelerated the development of cardiomyocytes hypertrophy partially via directly regulation of FoxO3-calcineurin pathway.

The narrow leaf1 (nal1) mutant of rice (Oryza sativa L.) exhibits a narrow leaf phenotype. Previous studies have shown that NAL1 modulates leaf size by affecting vein patterning and cell division; however, the underlying mechanism remains unclear. Here, we report that the nal1 mutant shows reduced size of the leaf abaxial epidermal cells and culm parenchyma cells compared with the wild type (WT), indicating that NAL1 also regulates cell expansion. To understand the molecular mechanism of the reduced cell size phenotype, leaves of 40-day-old nal1 mutant and WT seedlings were subjected to RNA-Seq analysis, which has identified 4277 differentially expressed genes (DEGs) between WT and the nal1 mutant. Gene ontology (GO) enrichment analysis revealed a large number of genes down-regulated in the nal1 mutant were involved in cell wall formation. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that NAL1-regulated DEGs, such as ARFs and SAURs, were mapped in auxin signal transduction and auxin-regulated cell expansion pathways. A combination of RNA-Seq analysis and gene expression validation using RT-qPCR suggested that NAL1 is involved in the regulation of auxin-mediated acid growth in rice. These results indicate that, in addition to controlling cell division, NAL1 controls leaf width, at least partially, through its effect on cell expansion, probably via the acid growth mechanism.

Tendon stem/progenitor cells (TSPC) are potential targets for regenerative medicine and the treatment of tendon injuries. The frequency of such injuries increases in elderly patients while the proportion of functional TSPCs in tendon tissue decreases, protracting tendon repair. Using atomic force microscopy (AFM), we show that cell stiffness and size increase in TSPCs isolated from elderly patients (A-TSPC) compared to TSPCs from younger patients (Y-TSPC). Additionally, two-photon excited fluorescence (TPEF) microscopy revealed a denser, well-structured actin cytoskeleton in A-TSPC, which correlates with the augmented cell stiffness. Treating A-TSPC with ROCK-inhibitor, reverses these age-related changes, and has rejuvenating effect on cell morphology and stiffness. We assume that cellular stiffness is a suitable marker for cell aging and ROCK a potential target for therapeutic applications of cell rejuvenation.

The importance of the biophysical cellular environment in cancer development has been increasingly recognised but so far has been only superficially studied. Here we investigated the effect of cell-like substrate topography on ovarian cancer cell behaviour and potential underlying signalling pathways. We observed changes in cell morphology in response to substrate topography, which implies modification of structure-function associations. Differences in focal adhesion signalling and Rho/ROCK activity suggested their involvement in the biomechanically-driven cellular responses. Cell-like topography was also shown to modulate the MAPK pathway and hence potentially regulate cell proliferation. The selective regulation of the cells by the mechanotransduction pathways that we noted has wide ranging implications for understanding cancer development. We established that the physical architecture of cell culture substrate is sufficient to influence cancer cell behaviour, independent of genetic composition or biochemical milieu.

The majority of studies on possible roles for collagen hydrolysates in human health have focused on their effects on bone and skin. Hydroxyprolyl-glycine (Hyp-Gly) was recently identified as a novel collagen hydrolysate-derived dipeptide in human blood. However, any possible health benefits of Hyp-Gly remain unclear. Here, we report the effects of Hyp-Gly on differentiation and hypertrophy of murine skeletal muscle C2C12 cells. Hyp-Gly increased the fusion index, the myotube size, and the expression of the myotube-specific myosin heavy chain (MyHC) and tropomyosin structural proteins. Hyp-Gly increased the phosphorylation of Akt, mTOR, and p70S6K in myoblasts, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited their phosphorylation by Hyp-Gly. LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin repressed the enhancing effects of Hyp-Gly on MyHC and tropomyosin expression. The peptide/histidine transporter 1 (PHT1) was highly expressed in both myoblasts and myotubes, and co-administration of histidine inhibited Hyp-Gly-induced phosphorylation of p70S6K in myoblasts and myotubes. These results indicate that Hyp-Gly can induce myogenic differentiation and myotube hypertrophy and suggest that Hyp-Gly promotes myogenic differentiation by activating the PI3K/Akt/mTOR signaling pathway, perhaps depending on PHT1 for entry into cells.

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities.

In this work, we demonstrated for the first time that S-propargyl-cysteine (SPRC, also named as ZYZ-802), a novel hydrogen sulfide (H2S)-releasing compound, had renoprotective effects on streptozotocin (STZ)-induced diabetic kidney injury. SPRC treatment significantly reduced the level of creatinine, kidney to body weight ratio and in particular, markedly decreased 24-h urine microalbuminuria excretion. SPRC suppressed the mRNA expression of fibronectin and type IV collagen. In vitro, SPRC inhibited mesangial cells over-proliferation and hypertrophy induced by high glucose. Additionally, SPRC attenuated inflammation in diabetic kidneys. SPRC also reduced transforming growth factor β1 (TGF-β1) signaling and expression of phosphorylated Smad3 (p-Smad3) pathway. Moreover, SPRC inhibited phosphorylation of ERK, p38 protein. Taken together, SPRC was demonstrated to be a potential therapeutic candidate to suppress diabetic nephropathy.

Elongation of endothelial cells is an important process in vascular formation and is expected to be a therapeutic target for inhibiting tumor angiogenesis. We have previously demonstrated that inhibition of mTORC1 and mTORC2 impaired endothelial cell elongation, although the mechanism has not been well defined. In this study, we analyzed the effects of the mTORC1-specific inhibitor everolimus and the mTORC1/mTORC2 dual inhibitor KU0063794 on the cytoskeletal organization and morphology of endothelial cell lines. While both inhibitors equally inhibited cell proliferation, KU0063794 specifically caused abnormal accumulation of F-actin and disordered distribution of microtubules, thereby markedly impairing endothelial cell elongation and tube formation. The effects of KU0063794 were phenocopied by paclitaxel treatment, suggesting that KU0063794 might impair endothelial cell morphology through over-stabilization of microtubules. Although mTORC1 is a key signaling molecule in cell proliferation and has been considered a target for preventing angiogenesis, mTORC1 inhibitors have not been sufficient to suppress angiogenesis. Our results suggest that mTORC1/mTORC2 dual inhibition is more effective for anti-angiogenic therapy, as it impairs not only endothelial cell proliferation, but also endothelial cell elongation.

Primary cilium is a microtubule-based non-motile organelle that plays critical roles in kidney pathophysiology. Our previous studies revealed that the lengths of primary cilia decreased upon renal ischemia/reperfusion injury and oxidative stress, and restored with recovery. Here, we tested the hypothesis that lack of primary cilium causes epithelial to mesenchymal transition (EMT) of kidney tubule cells. We investigated the alteration of length of primary cilia in TGF-β-induced EMT via visualization of primary cilia by fluorescence staining against acetylated α-tubulin. EMT was determined by measuring mesenchymal protein expression using quantitative PCR and indirect fluorescence staining. As a result, TGF-β treatment decreased ciliary length along with EMT. To test whether defect of primary cilia trigger onset of EMT, cilia formation was disturbed by knock down of ciliary protein using siRNA along with/without TGF-β treatment. Knock down of Arl13b and Ift20 reduced cilia elongation and increased expression of EMT markers such as fibronectin, α-SMA, and collagen III. TGF-β-induced EMT was greater as well in Arl13b and Ift20-knock down cells compared to control cells. Taken together, deficiency of primary cilia trigger EMT and exacerbates it under pro-fibrotic signals.

Tamoxifen is a selective estrogen receptor (ER) modulator which is widely used to generate inducible conditional transgenic mouse models. Activation of ER signaling plays an important role in the regulation of adipose tissue (AT) metabolism. We therefore tested the hypothesis that tamoxifen administration causes changes in AT biology in vivo. 12 weeks old male C57BL/6NTac mice were treated with either tamoxifen (n = 18) or vehicle (n = 18) for 5 consecutive days. Tamoxifen treatment effects on body composition, energy homeostasis, parameters of AT biology, glucose and lipid metabolism were investigated up to an age of 18 weeks. We found that tamoxifen treatment causes: I) significantly increased HbA1c, triglyceride and free fatty acid serum concentrations (p 0.01), II) browning of subcutaneous AT and increased UCP-1 expression, III) increased AT proliferation marker Ki67 mRNA expression, IV) changes in adipocyte size distribution, and V) transient body composition changes. Tamoxifen may induce changes in body composition, whole body glucose and lipid metabolism and has significant effects on AT biology, which need to be considered when using Tamoxifen as a tool to induce conditional transgenic mouse models. Our data further suggest that tamoxifen-treated wildtype mice should be characterized in parallel to experimental transgenic models to control for tamoxifen administration effects.

Synaptotagmin-like protein 2-a (Slp2-a) was originally described as a membrane trafficking protein that consists of a Slp homology domain (SHD), a linker domain, and tandem C2 domains (named the C2A domain and C2B domain). Slp2-a mediates docking of Rab27-bearing vesicles to the plasma membrane through simultaneous interaction with Rab27 and phospholipids in the plasma membrane. We have recently reported that Slp2-a regulates renal epithelial cell size through interaction with Rap1GAP2 via the C2B domain independently of Rab27 and demonstrated the presence of excess activation of ezrin, a membrane-cytoskeleton linker and signal transducer, in Slp2-a-knockdown Madin–Darby canine kidney II (MDCK II) cells. However, the precise mechanism of ezrin inactivation by Slp2-a in cell size control has remained largely unknown. In this study, we investigated the functional relationship between Slp2-a and ezrin in MDCK II cells. The results showed that activation of ezrin in control MDCK II cells either pharmacologically or by overexpression of a constitutively active ezrin mutant caused an increase in cell size, whereas inactivation of ezrin in Slp2-a-knockdown cells by a specific ezrin inhibitor restored them to their normal cell size. We also found that Slp2-a interacts via its previously uncharacterized linker domain with protein phosphatase 1β (PP1β), which inactivates ezrin, and that the interaction is required for the plasma membrane localization of PP1β. These results indicate that Slp2-a inactivates ezrin by recruiting PP1 to the plasma membrane.

Cardiac hypertrophy is determined by an increase of cell size in cardiomyocytes (CMCs). Among the cellular processes regulating the growth of cell size, the increase of protein synthesis rate represents a critical event. Most of translational factors promoting protein synthesis stimulate cardiac hypertrophy. In contrast, activity of translational repressor factors, in cardiac hypertrophy, is not fully determined yet. Here we report the effect of a translational modulator, eIF6/p27BBP in the hypertrophy of neonatal rat CMCs. The increase of eIF6 levels surprisingly prevent the growth of cell size induced by phenylephrine, through a block of protein synthesis without affecting skeletal rearrangement and ANF mRNA expression. Thus, this work uncovers a new translational cardiac regulator independent by other well-known factors such as mTOR signalling or eIF2β.

Protein kinase B2 (AKT2) is implicated in diverse process of cardiomyocyte signaling including survival and metabolism. However, the role of AKT2 in myocardium development and the signaling pathway is rarely understood. Therefore, we sought to determine the effect of AKT2 deletion on heart development and its downstream targets. By using experimental animal models and neonatal rat cardiomyocytes (NRCMs), we observed that AKT2 deficiency induces retardation of heart development and increased systemic blood pressure (BP) without affecting cardiac function. Further investigation suggested that deficiency of AKT2 in myocardium results in diminished MEF2A abundance, which induced decreased size of cardiomyocytes. We additionally confirmed that EndoG, which is also regulated by AKT2, is a suppressor of MEF2A in myocardium. Finally, our results proved that AKT2 deficiency impairs the response to β-adrenergic stimuli that normally causes hypertrophy in cardiomyocytes by downregulating MEF2A expression. Our data are the first to show the important role of AKT2 in determining the size of myocardium, its deficiency causes retardation of cardiomyocyte development. We also proved a novel pathway of heart development involving EndoG and MEF2A regulated by AKT2.

The capacity of liver regeneration is critical for patients with liver diseases. However, cellular and molecular mechanisms of liver regeneration are still incompletely defined. Here, we assessed roles of LASS2 in liver regeneration following partial hepatectomy (PHx) in mice. Our results showed that protein level of LASS2 remarkably increased during liver regeneration after PHx in wildtype (WT) mice. Comparing to WT mice, liver regeneration index after PHx was significantly decreased from day 1 to day 5 in liver-specific LASS2 knockout (LASS2-LKO) mice. Interestingly, liver mass of LASS2-LKO mice could sufficiently recover at day 14 after PHx. Immunohistochemistry (IHC) and western blot analyses revealed that proliferation markers, such as PCNA and Ki67, were potently reduced during liver regeneration in LASS2-LKO mice. In addition, several cell cycle related molecules, such as cyclin A, CDK2 and p-Rb, were decreased in LASS2-LKO mice after PHx. Co-immunoprecipitation assay further revealed a decreased formation of CDK4/cyclin D1 complex after PHx in LASS2-LKO mice. However, phosphorylation of Akt was significantly activated from day 2 after PHx in LASS2-LKO mice when compared with that in WT mice, which may explain the recovery of liver mass at the late stage of liver regeneration in LASS2-LKO mice. Taken together, we conclude that LASS2 plays an important role in efficient liver regeneration in response to PHx.

Changes in extracellular osmolarity lead to alteration in cellular volume. In the study, we examined the effects of hyperosmolarity on short-circuit currents (Isc) in the rat ileum using the Ussing chamber technique. Mucosal exposure to 20 mM glucose evoked a decrease of ISC in the rat ileum, which was antagonized by the stretch-activated channel blocker GdCl3, TTX and atropine, respectively. In contrast, it was not blocked by phlorizin, a Na+-glucose cotransporter SGLT1 inhibitor. Furthermore, the unabsorbed substances, such as sucrose, lactulose or urea, also induced a decrease of ISC in rat ileum. ELISA results revealed that 20 mM glucose stimulated the release of histamine from rat ileum mucosa, which was attenuated by TTX. In addition, the glucose-induced ISC was depressed by pyrilamine, a histamine H1 receptor blocker (H1 antagonist) whereas it was not affected by ranitidine (H2 antagonist), clobenpropit (H3 antagonists) or JNJ7777120 (H4 antagonist), respectively. The ion substitution experiments suggest that the changes of Na+ and HCO3− ion flux underlie the glucose-induced ISC. In conclusion, osmotic stimulus decreased the basal ISC of rat ileum by evoking histamine release from ileum mucosa. The changes of Na+ and HCO3− ion transport are involved in the glucose-evoked decrease of basal ISC.

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell-substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis in Dictyostelium.

In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancer cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the (3)H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.

The histone methyltransferase Setdb1 represses gene expression by catalyzing lysine 9 of histone H3 trimethylation. Given that the conventional knockout of Setdb1 is embryo-lethal at the implantation stage, its role in craniofacial development is poorly understood. Here, we investigated the role of Setdb1, using conditional knockout mice-in which Setdb1 was deleted in the Meckel's cartilage (Setdb1 CKO)-and the mouse chondrogenic cell line ATDC5-in which Setdb1 was inhibited by siRNA. Deletion of Setdb1 in Meckel's cartilage, the supportive tissue in the embryonic mandible, led to its enlargement, instead of the degeneration that normally occurs. Chondrocytes from the Meckel's cartilage of Setdb1 CKO mice showed increased size. Furthermore, at embryonic days 16.5 and 18.5, part of the perichondrium was disrupted and mineralization was observed in the Meckel's cartilage. Proliferation analysis showed that inhibition of Setdb1 caused increased proliferation in chondrocytes in the Meckel's cartilage as well as in ATDC5 cells. Quantitative RT-PCR showed decreased expression of chondrogenic genes, such as Sox9, Mmp13, Collagen II, and Aggrecan, as a result of Setdb1 inhibition in ATDC5 cells. Along with these phenomenons, SMAD-dependent BMP signaling was significantly increased by the loss of Setdb1 in both the Meckel's cartilage of Setdb1 CKO mice and ATDC5 cells. Therefore, the abnormal development of Meckel's cartilage in Setdb1 CKO mice is partly due to the enhanced SMAD-dependent BMP signaling. Overall, to our knowledge, the present study is the first to show that epigenetic regulation by Setdb1 is indispensable for the embryonic development of Meckel's cartilage.

Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA-) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum.

Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds.

The Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/β-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active β-catenin associated with significant increases in protein content and gene expression of β-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/β-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM.

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

The position of the mitotic spindle plays a key role in spatial control of cell division. It is generally believed that when a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are usually unequal in size due to eccentric cleavage of the mother cell. Molecular mechanisms underlying the generation of unequal sized daughter cells have been extensively studied in Drosophila neuroblast and Caenorhabditis elegans zygote where the Gα subunit of the heterotrimeric G proteins and its binding partner - Pins in Drosophila and GPR-1/2 in C. elegans - are shown to be critical in governing spindle positioning and asymmetric cleavage of the mother cell. In mammalian system, although Gα and LGN (mammalian Pins homolog) are also required for spindle orientation, whether they can mediate asymmetric spindle positioning or asymmetric cleavage of the mother cell is not known. Here, by artificially targeting Gαi to the apical cortex in 3-D cultured MDCK cells, we established a system where asymmetric spindle positioning can be consistently induced. Interestingly, this asymmetrically positioned spindle does not lead to asymmetric cleavage; instead it results in equal sized daughter cells. Live cell time-lapse analysis revealed that anaphase spindle elongation compensated the original asymmetric spindle positioning. Our findings demonstrate that asymmetric spindle positioning does not necessarily lead to unequal sized daughter cells in mammalian system. We discuss potential mechanisms in generating unequal sized daughter cells.

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H(2)O(2))-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H(2)O(2) activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H(2)O(2) elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1h and induced apoptosis of most PC12 cells tested in 24h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H(2)O(2)-induced high membrane permeability and cell shrinkage, suppressed H(2)O(2)-activated chloride currents and protected PC12 cells from apoptosis induced by H(2)O(2). The results suggest that chloride channels may contribute to H(2)O(2)-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain-deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.

AMP-activated protein kinase (AMPK) is known as an important cellular energy sensor, but its in vivo role has not been fully understood. Recent studies provided surprising results that AMPK regulates cell polarity and mitosis under the control of tumour suppressor LKB1. Moreover, these newly found in vivo functions of AMPK are regulated by energy status in a cell autonomous manner. These findings provide novel insights into the physiological function of AMPK and the treatment of AMPK-related diseases such as cancer and diabetes.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.

Oligodendrocyte (OL) plays a critical role in myelination and axon maintenance in central nervous system. Recent studies show that OL can also express NMDA receptors in development and pathological situations in white matter. There is still lack of studies about OL properties and function in gray matter of brain. Here we reported that some glial cells in CA1 region of rat hippocampal slices (P15-23) had distinct electrophysiological characteristics from the other glia cells in this region, while they displayed uniform properties with OL from white matter in previous report; therefore, they were considered as OL in hippocampus. By loading dye in recording pipette and imaging with two-photon laser scanning microscopy, we acquired the high spatial resolution, three-dimension images of these special cells in live slices. The OL in hippocampus shows a complex process-bearing shape and the distribution of several processes is parallel to Schaffer fiber in CA1 region. When stimulating Schaffer fiber, OL displays a long duration depolarization mediated by inward rectifier potassium channel. This suggested that the OL in CA1 region could sense the neuronal activity and contribute to potassium clearance.

The morphology and function of Leydig cells are changed during the development, mature and senility of Leydig cells along the life span of males. This study was to observe the growth morphology of adult mouse Leydig cells in culture, aiming to provide a reference for furthermore understanding of the biological function of Leydig cells by in vitro model. Testes of two-month-old mice were decapsulated and then the Leydig cells were isolated by collagenase digestion and were cultured in DMEM/F12 supplemented with 10% FBS. The Leydig cells were identified by HSD3B staining and RT-PCR. After 48-h Leydig cell culture, both the nucleus and the cytoplasm were very clear under the optical microscope. The nucleus was big and round and the cytoplasm was filled with abundant lipid drops with a strong refractivity. After 5-day culture, Leydig cells were fully elongated in spindle, triangular, polygonal, oval or irregular shapes. Some cells grew in aggregation, and some cells grew independently. Leydig cells in aggregation elongated many cellular tentacles for intercellular connections, which formed an epithelium-like appearance. After HSD3B staining, the individual Leydig cells were stained with different extents, demonstrated that the heterogeneity of HSD3B activity in individual Leydig cells in primary culture. RT-PCR results showed that Leydig cells in culture after 5 days could express Leydig cell-specific transcriptions, HSD3B6, CYP17A1 and StAR. These results showed the morphological characterization of adult mouse Leydig cells in culture, which will lay a foundation to elucidate the relationship between the morphology and function of Leydig cells.